JP2021147426A - Protein molding and method for producing the same - Google Patents

Abstract

To provide a protein molding having excellent productivity and an advantageous production method therefor.SOLUTION: The present disclosure provides a molding of a composition including crosslinked protein, the crosslinked protein being a protein crosslinked with a linker represented by formula (1), (2) or (3), where R is a hydrogen atom or an organic group, and L is a divalent organic group.SELECTED DRAWING: None

Description

The present invention relates to a protein molded product and a method for producing the same.

In recent years, due to the growing awareness of environmental conservation, studies on alternative substances for petroleum-derived materials have been promoted, and proteins have been listed as candidates in terms of strength and the like. The protein is used as a molded product by heat and pressure molding, and is used as a protein fiber by a spinning operation. For example, Patent Document 1 discloses an animal fiber molded product having high mechanical properties such as stress-strain characteristics obtained by compression molding an aggregate of animal fibers. Recently, with the development of protein synthesis technology, the materialization of industrially producible artificial proteins has been studied. Among them, spider silk protein (spider silk fibroin) has the characteristics of being excellent in strength and elongation, and research on artificial proteins utilizing these characteristics is being studied.

The physical characteristics of a protein can be changed by subjecting it to chemical modification such as cross-linking. However, it is difficult to carry out the protein cross-linking step and the protein molding step at the same time, and these steps are usually carried out separately, which leads to an increase in the manufacturing burden. For example, in the case of producing a heat-pressurized molded product of a crosslinked protein, a crosslinked structure is introduced into the raw material protein by mixing the protein with a crosslinking agent such as isocyanate and heating, and then the obtained crosslinked protein is heated and pressurized. Mold. This is because isocyanate has high reactivity, so that cross-linking may proceed before the temperature required for molding is reached, which may lead to deterioration of the quality of the molded product. Further, in the case of producing cross-linked protein fibers, when a cross-linking agent such as isocyanate is added to a protein solution (dope solution) and heated (pre-spinning cross-linking), the draw ratio decreases in the subsequent drawing step to obtain desired physical properties. On the other hand, if the protein fiber after spinning is immersed in a cross-linking agent solution such as glutaraldehyde and heated (cross-linking after spinning), the reproducibility of cross-linking may decrease.

JP-A-2017-110132

In view of the above circumstances, an object of the present invention is to provide a protein molded product having excellent productivity and an advantageous production method thereof. Another object of the present invention is to provide artificial leather having excellent productivity.

When a composition containing a protein and an α, β-unsaturated carbonyl compound is used, the present inventors do not allow cross-linking to proceed at low temperatures, and the conditions under heating and pressurization during molding, and during drying after spinning or film casting. It was found that protein cross-linking proceeds well at heating temperature.

［式中、Ｌは２価の有機基を示す。］
［２］
架橋タンパク質を含む組成物の成形体であって、
前記架橋タンパク質が、式（２）又は（３）で表されるリンカーによって架橋されたタンパク質である、成形体。

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］
［３］
前記タンパク質が人工タンパク質である、［１］又は［２］に記載の成形体。
［４］
前記人工タンパク質が人工構造タンパク質である、［３］に記載の成形体。
［５］
前記人工構造タンパク質が人工フィブロインである、［４］に記載の成形体。
［６］
前記人工フィブロインが人工クモ糸フィブロインである、［５］に記載の成形体。
［７］
フィルム又は繊維の形態である、［１］〜［６］の何れかに記載の成形体。
［８］
見かけ上均一相である、［１］〜［７］の何れかに記載の成形体。
［９］
架橋タンパク質を含む組成物の成形体の製造方法であって、
タンパク質及び式（１ａ）で表される化合物を含む組成物を加熱する工程を含む、成形体の製造方法。

［式中、Ｌは２価の有機基を示す。］

［式中、Ｌは２価の有機基を示す。］
［１０］
前記成形体が繊維の形態であり、
前記加熱が、乾式紡糸、湿式紡糸及び乾湿式紡糸のうちの何れか一種により得られた未乾燥繊維を乾燥するための加熱である、［８］に記載の成形体の製造方法。
［１１］
前記成形体がフィルムの形態であり、
前記加熱が、キャスト成形により得られた未乾燥フィルムを乾燥するため、又はアニーリング処理のための加熱である、［９］に記載の成形体の製造方法。
［１２］
前記組成物を加熱と同時に加圧する、［９］に記載の成形体の製造方法。
［１３］
前記成形体が見かけ上均一相である、［９］〜［１２］のいずれかに記載の成形体の製造方法。
［１４］
架橋タンパク質を含む組成物の成形体の製造方法であって、
タンパク質と、式（２ａ）、（２ｂ）及び（３ａ）で表される化合物からなる群より選択される少なくとも一種と、を含む組成物を加熱する工程を含む、成形体の製造方法。

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］
［１５］
前記成形体が繊維の形態であり、
前記加熱が、乾式紡糸、湿式紡糸及び乾湿式紡糸のうちの何れか一種により得られた未乾燥繊維を乾燥するための加熱である、［１４］に記載の成形体の製造方法。
［１６］
前記成形体がフィルムの形態であり、
前記加熱が、キャスト成形により得られた未乾燥フィルムを乾燥するため、又はアニーリング処理のための加熱である、［１４］に記載の成形体の製造方法。
［１７］
前記組成物を加熱と同時に加圧する、［１４］に記載の成形体の製造方法。
［１８］
前記成形体が見かけ上均一相である、［１４］〜［１７］のいずれかに記載の成形体の製造方法。
［１９］
前記タンパク質が人工タンパク質である、［９］〜［１８］の何れかに記載の成形体の製造方法。
［２０］
前記人工タンパク質が人工構造タンパク質である、［１９］に記載の成形体の製造方法。
［２１］
前記人工構造タンパク質が人工フィブロインである、［２０］に記載の成形体の製造方法。
［２２］
前記人工フィブロインが人工クモ糸フィブロインである、［２１］に記載の成形体の造方法。
［２３］
基材層と、該基材層上に接合された表皮層とを備え、
該表皮層が、タンパク質が式（１）で表されるリンカーによって架橋された架橋タンパク質を含む成形体を含む、人造皮革。

［式中、Ｌは２価の有機基を示す。］
［２４］
基材層と、該基材層上に接合された表皮層とを備え、
該表皮層が、タンパク質が式（２）又は（３）で表されるリンカーによって架橋された架橋タンパク質を含む成形体を含む、人造皮革。

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］
［２５］
基材層が、編布、織布、及び不織布のうちの何れかを含む基布である、［２３］又は［２４］に記載の人造皮革。
［２６］
基材層と、該基材層上に接合された表皮層とを備え、
前記基材層が、編布又は織布を含む基布であり、
該基布が、タンパク質が式（１）で表されるリンカーによって架橋された、繊維状の架橋タンパク質を含む成形体を含む、人造皮革。

［式中、Ｌは２価の有機基を示す。］
［２７］
基材層と、該基材層上に接合された表皮層とを備え、
前記基材層が、編布又は織布を含む基布であり、
該基布が、タンパク質が式（２）又は（３）で表されるリンカーによって架橋された、繊維状の架橋タンパク質を含む成形体を含む、人造皮革。

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］
［２８］
ポリマー組成物が含浸された不織布を含む基材層を備え、
前記不織布が、タンパク質が式（１）で表されるリンカーによって架橋された、繊維状の架橋タンパク質を含む成形体を含む、人造皮革。

［式中、Ｌは２価の有機基を示す。］
［２９］
ポリマー組成物が含浸された不織布を含む基材層を備え、
前記不織布が、タンパク質が式（２）又は（３）で表されるリンカーによって架橋された、繊維状の架橋タンパク質を含む成形体を含む、人造皮革。

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］
［３０］
ポリマー組成物が含浸された不織布を含む基材層を備え、
前記ポリマー組成物が、タンパク質が式（１）で表されるリンカーによって架橋された架橋タンパク質を含む成形体を含有する、人造皮革。

［式中、Ｌは２価の有機基を示す。］
［３１］
ポリマー組成物が含浸された不織布を含む基材層を備え、
前記ポリマー組成物が、タンパク質が式（２）又は（３）で表されるリンカーによって架橋された架橋タンパク質を含む成形体を含有する、人造皮革。

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］
［３２］
前記タンパク質が人工タンパク質である、［２３］〜［３１］の何れかに記載の人造皮革。
［３３］
前記人工タンパク質が人工構造タンパク質である、［３２］に記載の人造皮革。
［３４］
前記人工構造タンパク質が人工フィブロインである、［３３］に記載の人造皮革。
［３５］
前記人工フィブロインが人工クモ糸フィブロインである、［３４］に記載の人造皮革。
［３６］
基材層と、該基材層上に接合された表皮層とを備える人造皮革の製造方法であって、
タンパク質と、式（１ａ）で表される化合物と、を含む組成物を展延して表皮層前駆体を形成する工程と、
前記表皮層前駆体を加熱する工程と、を含み、
前記表皮層は、前記タンパク質が式（１）で表されるリンカーによって架橋された架橋タンパク質を含有する、人造皮革の製造方法。

［式中、Ｌは２価の有機基を示す。］

［式中、Ｌは２価の有機基を示す。］
［３７］
基材層と、該基材層上に接合された表皮層とを備える人造皮革の製造方法であって、
タンパク質と、式（２ａ）、（２ｂ）及び（３ａ）で表される化合物からなる群より選択される少なくとも一種と、を含む組成物を展延して表皮層前駆体を形成する工程と、
前記表皮層前駆体を加熱する工程と、を含み、
前記表皮層は、前記タンパク質が式（２）で表されるリンカーによって架橋された架橋タンパク質を含有する、人造皮革の製造方法。

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］
［３８］
基材層と、該基材層上に接合された表皮層とを備える人造皮革の製造方法であって、
架橋タンパク質を含むタンパク質繊維を編成又は織成して前記基材層を得る工程を含み、
前記架橋タンパク質は、タンパク質が式（１）で表されるリンカーによって架橋されたものである、人造皮革の製造方法。

［式中、Ｌは２価の有機基を示す。］
［３９］
基材層と、該基材層上に接合された表皮層とを備えた人造皮革の製造方法であって、
架橋タンパク質を含むタンパク質繊維を編成又は織成して前記基材層を得る工程を含み、
前記架橋タンパク質は、タンパク質が式（２）又は（３）で表されるリンカーによって架橋されたものである、人造皮革の製造方法。

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］
［４０］
基材層を備える人造皮革の製造方法であって、
架橋タンパク質を含むポリマー組成物を不織布に含浸させて基剤層を得る工程を含み、
前記架橋タンパク質は、タンパク質が式（１）で表されるリンカーによって架橋されたものである、人造皮革の製造方法。

［式中、Ｌは２価の有機基を示す。］
［４１］
基材層を備える人造皮革の製造方法であって、
架橋タンパク質を含むタンパク質繊維を不織布に含浸させて基剤層を得る工程を含み、
前記架橋タンパク質は、タンパク質が式（２）又は（３）で表されるリンカーによって架橋されたものである、人造皮革の製造方法。

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］
［４２］
基材層を備える人造皮革の製造方法であって、
タンパク質と、式（１ａ）で表される化合物と、を含む組成物を不織布に含浸させた状態で加熱して基剤層を得る工程を含み、
前記基剤層が、タンパク質が式（１）で表されるリンカーによって架橋された架橋タンパク質を含む成形体が含浸された不織布を含む、人造皮革の製造方法。

［式中、Ｌは２価の有機基を示す。］

［式中、Ｌは２価の有機基を示す。］
［４３］
基材層を備える人造皮革の製造方法であって、
タンパク質と、式（２ａ）、（２ｂ）及び（３ａ）で表される化合物からなる群より選択される少なくとも一種と、を含む組成物を不織布に含浸させた状態で加熱して基剤層を得る工程を含み、
前記基剤層が、タンパク質が式（２）で表されるリンカーによって架橋された架橋タンパク質を含む成形体が含浸された不織布を含む、人造皮革の製造方法。

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］
［４４］
前記タンパク質が人工タンパク質である、［３６］〜［４３］の何れかに記載の人造皮革の製造方法。
［４５］
前記人工タンパク質が人工構造タンパク質である、［４４］に記載の人造皮革の製造方法。
［４６］
前記人工構造タンパク質が人工フィブロインである、［４５］に記載の人造皮革の製造方法。
［４７］
前記人工フィブロインが人工クモ糸フィブロインである、［４６］に記載の人造皮革の製造方法。 The present invention provides the following [1] to [47].
[1]
A molded product of a composition containing a crosslinked protein.
A molded product in which the crosslinked protein is a protein crosslinked by a linker represented by the formula (1).

[In the formula, L represents a divalent organic group. ]
[2]
A molded product of a composition containing a crosslinked protein.
A molded product in which the crosslinked protein is a protein crosslinked by a linker represented by the formula (2) or (3).

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]
[3]
The molded article according to [1] or [2], wherein the protein is an artificial protein.
[4]
The molded product according to [3], wherein the artificial protein is an artificial structural protein.
[5]
The molded article according to [4], wherein the artificial structural protein is artificial fibroin.
[6]
The molded product according to [5], wherein the artificial fibroin is an artificial spider silk fibroin.
[7]
The molded product according to any one of [1] to [6], which is in the form of a film or a fiber.
[8]
The molded product according to any one of [1] to [7], which is apparently a uniform phase.
[9]
A method for producing a molded product of a composition containing a crosslinked protein.
A method for producing a molded product, which comprises a step of heating a composition containing a protein and a compound represented by the formula (1a).

[In the formula, L represents a divalent organic group. ]

[In the formula, L represents a divalent organic group. ]
[10]
The molded product is in the form of fibers.
The method for producing a molded product according to [8], wherein the heating is heating for drying undried fibers obtained by any one of dry spinning, wet spinning and dry wet spinning.
[11]
The molded product is in the form of a film.
The method for producing a molded product according to [9], wherein the heating is heating for drying an undried film obtained by cast molding or for an annealing treatment.
[12]
The method for producing a molded product according to [9], wherein the composition is pressurized at the same time as heating.
[13]
The method for producing a molded product according to any one of [9] to [12], wherein the molded product has an apparently uniform phase.
[14]
A method for producing a molded product of a composition containing a crosslinked protein.
A method for producing a molded product, which comprises a step of heating a composition containing a protein and at least one selected from the group consisting of compounds represented by the formulas (2a), (2b) and (3a).

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]
[15]
The molded product is in the form of fibers.
The method for producing a molded product according to [14], wherein the heating is heating for drying undried fibers obtained by any one of dry spinning, wet spinning and dry wet spinning.
[16]
The molded product is in the form of a film.
The method for producing a molded product according to [14], wherein the heating is heating for drying an undried film obtained by cast molding or for an annealing treatment.
[17]
The method for producing a molded product according to [14], wherein the composition is pressurized at the same time as heating.
[18]
The method for producing a molded product according to any one of [14] to [17], wherein the molded product has an apparently uniform phase.
[19]
The method for producing a molded product according to any one of [9] to [18], wherein the protein is an artificial protein.
[20]
The method for producing a molded product according to [19], wherein the artificial protein is an artificial structural protein.
[21]
The method for producing a molded product according to [20], wherein the artificial structural protein is artificial fibroin.
[22]
The method for producing a molded product according to [21], wherein the artificial fibroin is artificial spider silk fibroin.
[23]
A base material layer and an epidermis layer bonded onto the base material layer are provided.
An artificial leather in which the epidermis layer contains a molded product containing a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1).

[In the formula, L represents a divalent organic group. ]
[24]
A base material layer and an epidermis layer bonded onto the base material layer are provided.
An artificial leather in which the epidermis layer contains a molded product containing a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (2) or (3).

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]
[25]
The artificial leather according to [23] or [24], wherein the base material is a base fabric containing any of a knitted fabric, a woven fabric, and a non-woven fabric.
[26]
A base material layer and an epidermis layer bonded onto the base material layer are provided.
The base material layer is a base fabric containing a knitted fabric or a woven fabric.
An artificial leather in which the base fabric contains a molded product containing a fibrous crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1).

[In the formula, L represents a divalent organic group. ]
[27]
A base material layer and an epidermis layer bonded onto the base material layer are provided.
The base material layer is a base fabric containing a knitted fabric or a woven fabric.
An artificial leather in which the base fabric contains a molded product containing a fibrous crosslinked protein in which the protein is crosslinked by a linker represented by the formula (2) or (3).

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]
[28]
It comprises a substrate layer containing a non-woven fabric impregnated with a polymer composition.
An artificial leather in which the non-woven fabric contains a molded product containing a fibrous crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1).

[In the formula, L represents a divalent organic group. ]
[29]
It comprises a substrate layer containing a non-woven fabric impregnated with a polymer composition.
An artificial leather in which the non-woven fabric contains a molded product containing a fibrous crosslinked protein in which the protein is crosslinked by a linker represented by the formula (2) or (3).

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]
[30]
It comprises a substrate layer containing a non-woven fabric impregnated with a polymer composition.
An artificial leather in which the polymer composition contains a molded product containing a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1).

[In the formula, L represents a divalent organic group. ]
[31]
It comprises a substrate layer containing a non-woven fabric impregnated with a polymer composition.
An artificial leather in which the polymer composition contains a molded product containing a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (2) or (3).

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]
[32]
The artificial leather according to any one of [23] to [31], wherein the protein is an artificial protein.
[33]
The artificial leather according to [32], wherein the artificial protein is an artificial structural protein.
[34]
The artificial leather according to [33], wherein the artificial structural protein is artificial fibroin.
[35]
The artificial leather according to [34], wherein the artificial fibroin is an artificial spider silk fibroin.
[36]
A method for producing artificial leather including a base material layer and a skin layer bonded on the base material layer.
A step of spreading a composition containing a protein and a compound represented by the formula (1a) to form an epidermal precursor.
Including the step of heating the epidermis layer precursor.
A method for producing artificial leather, wherein the epidermis layer contains a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1).

[In the formula, L represents a divalent organic group. ]

[In the formula, L represents a divalent organic group. ]
[37]
A method for producing artificial leather including a base material layer and a skin layer bonded on the base material layer.
A step of spreading a composition containing a protein and at least one selected from the group consisting of compounds represented by the formulas (2a), (2b) and (3a) to form an epidermal precursor.
Including the step of heating the epidermis layer precursor.
A method for producing artificial leather, wherein the epidermis layer contains a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (2).

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]
[38]
A method for producing artificial leather including a base material layer and a skin layer bonded on the base material layer.
Including the step of knitting or weaving a protein fiber containing a crosslinked protein to obtain the base material layer.
The crosslinked protein is a method for producing artificial leather, in which the protein is crosslinked by a linker represented by the formula (1).

[In the formula, L represents a divalent organic group. ]
[39]
A method for producing artificial leather including a base material layer and a skin layer bonded on the base material layer.
Including the step of knitting or weaving a protein fiber containing a crosslinked protein to obtain the base material layer.
The crosslinked protein is a method for producing artificial leather, wherein the protein is crosslinked by a linker represented by the formula (2) or (3).

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]
[40]
A method for producing artificial leather having a base material layer.
A step of impregnating a non-woven fabric with a polymer composition containing a crosslinked protein to obtain a base layer is included.
The crosslinked protein is a method for producing artificial leather, in which the protein is crosslinked by a linker represented by the formula (1).

[In the formula, L represents a divalent organic group. ]
[41]
A method for producing artificial leather having a base material layer.
A step of impregnating a non-woven fabric with a protein fiber containing a crosslinked protein to obtain a base layer is included.
The crosslinked protein is a method for producing artificial leather, wherein the protein is crosslinked by a linker represented by the formula (2) or (3).

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]
[42]
A method for producing artificial leather having a base material layer.
A step of obtaining a base layer by heating a non-woven fabric in which a composition containing a protein and a compound represented by the formula (1a) is impregnated is included.
A method for producing artificial leather, wherein the base layer comprises a non-woven fabric impregnated with a molded product containing a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1).

[In the formula, L represents a divalent organic group. ]

[In the formula, L represents a divalent organic group. ]
[43]
A method for producing artificial leather having a base material layer.
The base layer is heated in a state where the non-woven fabric is impregnated with a composition containing a protein and at least one selected from the group consisting of compounds represented by the formulas (2a), (2b) and (3a). Including the process of obtaining
A method for producing artificial leather, wherein the base layer comprises a non-woven fabric impregnated with a molded product containing a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (2).

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]
[44]
The method for producing artificial leather according to any one of [36] to [43], wherein the protein is an artificial protein.
[45]
The method for producing artificial leather according to [44], wherein the artificial protein is an artificial structural protein.
[46]
The method for producing artificial leather according to [45], wherein the artificial structural protein is artificial fibroin.
[47]
The method for producing artificial leather according to [46], wherein the artificial fibroin is artificial spider silk fibroin.

In research on protein molded products, there is an attempt to increase the molecular weight of the raw material protein by cross-linking operation in order to further improve the physical properties. However, there is a concern that the addition of this cross-linking operation will cause an increase in manufacturing energy and thus a decrease in productivity.

According to the present invention, a protein can be sufficiently crosslinked by a step involving heating (eg, during drying, during pressure molding) in a series of manufacturing processes of a protein molded product (for example, artificial leather). That is, according to the present invention, it is not necessary to carry out a heating operation for crosslinking separately from the main steps in the manufacturing process, and at the heating temperature required for heat and pressure molding, or at the drying temperature of the fiber or film. The cross-linking reaction can proceed sufficiently. Therefore, the present invention is excellent in the productivity of protein molded products (for example, artificial leather).

Further, the obtained molded product is less likely to crack even in a wet state and may have water resistance. The artificial leather according to the present invention has various merits such as reduction of production energy as compared with the conventional artificial leather using chemical fiber, polyurethane resin or the like.

It is a schematic diagram which shows an example of the domain sequence of artificial fibroin. It is a figure which shows the distribution of the value of z / w (%) of naturally-derived fibroin. It is a figure which shows the distribution of the value of x / y (%) of naturally-derived fibroin. It is a schematic diagram which shows an example of the domain sequence of artificial fibroin. It is a schematic diagram which shows an example of the domain sequence of artificial fibroin. It is a schematic cross-sectional view of a pressure molding machine. (A) is a state before the composition is introduced, (b) is a state immediately after the composition is introduced, and (c) is a schematic cross-sectional view of a pressure molding machine in a state where the composition is heated and pressed. .. It is a schematic cross-sectional view of the artificial leather which concerns on one Embodiment of this invention, (a) is a schematic cross-sectional view of "artificial leather", and (b) is a schematic cross-sectional view of "synthetic leather". It is a graph which shows the result of the GPC test of Example 1 and Comparative Example 1. It is a photograph showing the result of the crack test of Example 1 (left) and Comparative Example 1 (right), (a) is a state before the test, (b) is a state when 140 hours have passed at 50 ° C., (c). ) Is a photograph showing the state when 192 hours have passed at 80 ° C. It is a graph which shows the result of the hygroscopicity test of Example 2 and Comparative Example 1. It is a schematic diagram of the manufacturing method of synthetic leather which concerns on one Embodiment. It is a schematic diagram of the manufacturing method of synthetic leather which concerns on one Embodiment. It is explanatory drawing of the electrospinning apparatus which concerns on one Embodiment. It is a schematic diagram which shows an example of the manufacturing apparatus at the time of manufacturing synthetic leather by the dry method. It is a schematic diagram which shows an example of the manufacturing apparatus at the time of manufacturing synthetic leather by the wet method.

The present invention will be described in more detail below.

［式中、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］ [First Embodiment]
The first embodiment of the present invention is a molded product of a composition containing a crosslinked protein, wherein the crosslinked protein is a protein crosslinked by a linker represented by the formula (1), (2) or (3). , A molded body. The compound represented by the formula (1), (2) or (3) is bound to the protein in the wavy line portion in the formula.

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

(protein)
The protein may be any protein that can react with the maleimide group of the compound represented by the formula (1a), (2a), (2b) or (3a), and examples thereof include natural proteins and artificial proteins. Examples of the artificial protein include any protein that can be produced on an industrial scale, and examples thereof include proteins that can be used for industrial purposes, proteins that can be used for medical purposes, and structural proteins. Specific examples of proteins that can be used for industrial or medical purposes include enzymes, regulatory proteins, receptors, peptide hormones, cytokines, membrane or transport proteins, antigens used for vaccination, vaccines, antigen-binding proteins, immunostimulatory proteins, etc. Examples include allergens, full-length antibodies or antibody fragments or derivatives. Specific examples of structural proteins include spider silk (spider silk), silk moth silk, keratin, collagen, elastin and resilin, and proteins derived from these.

Artificial proteins also include recombinant proteins and synthetic proteins. That is, as used herein, the term "artificial protein" means an artificially produced protein. The artificial protein may be a protein whose domain sequence is different from the amino acid sequence of the naturally occurring protein, or may be a protein having the same amino acid sequence as the naturally occurring protein. Further, the "artificial protein" may be one in which the amino acid sequence of the naturally occurring protein is used as it is, or one in which the amino acid sequence is modified based on the amino acid sequence of the naturally occurring protein (for example, cloned natural). It may be an amino acid sequence modified by modifying the gene sequence of a derived protein, or it may be artificially designed and synthesized regardless of a naturally occurring protein (for example, it encodes a designed amino acid sequence). It may have a desired amino acid sequence by chemically synthesizing a nucleic acid).

［式中、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］ As the protein to be used, artificial fibroin is preferable, and artificial spider silk fibroin is more preferable, because it is excellent in heat retention, hygroscopic heat generation and / or flame retardancy. When the protein is artificial fibroin (preferably artificial spider silk fibroin), the synthetic leather according to the present embodiment can be further imparted with heat retention, moisture absorption and heat generation and / or flame retardant properties, and the synthetic leather can be further imparted. The value as is higher. In addition, artificial fibroin has a high fiber-forming ability because it can be easily fibrilized. Therefore, it may be desired to improve productivity in artificial leather containing a knitted body or a non-woven fabric made of artificial fibers containing such artificial fibroin as a base material layer.

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

The artificial protein according to this embodiment may have 50 or more amino acid residues. The number of amino acid residues may be, for example, 100 or more or 150 or more, 200 or more or 250 or more, and preferably 300 or more, 350 or more, 400 or more, 450 or more or 500 or more.

(Artificial structural protein)
The artificial protein according to this embodiment may be, for example, a structural protein. The structural protein means a protein related to the structure of a living body, a protein constituting a structure produced by the living body, or a protein derived from them. Structural proteins also refer to proteins that self-aggregate under certain conditions to form structures such as fibers, films, resins, gels, micelles, and nanoparticles. In nature, for example, fibroin, keratin, and collagen. Examples include gen, elastin and resilin.

The structural protein may be an artificial structural protein. As used herein, the term "artificial structural protein" means an artificially produced structural protein. The artificially structured protein may be a polypeptide capable of reacting with the maleimide group of the compound represented by the formula (1a), (2a), (2b) or (3a). It may be a structural protein produced microbially by artificial gene, including an amino acid sequence improved from the viewpoint of moldability and productivity, and is not limited to the sequence of a naturally occurring structural protein.

When artificially molding an artificially structured protein, amino acids with smaller side chains are more likely to form hydrogen bonds, and it is easier to obtain a high-strength molded product. Further, since the alanine residue and the glycine residue are non-polar amino acids in the side chain, they are arranged so as to face inward in the folding process in polypeptide production, and easily form an α-helix structure or a β-sheet structure. Therefore, it is desirable that the proportion of amino acids such as glycine residue, alanine residue, and serine residue is high. For example, the alanine residue content may be 10-40%, 12-40%, 15-40%, 18-40%, 20-40%. It may be 22 to 40%. For example, the glycine residue content may be 10 to 55%, 11% to 55%, 13% to 55%, 15% to 55%, 18% to. It may be 55%, 20% to 55%, 22% to 55%, 25% to 55%.

In addition, in this specification, "alanine residue content" is a value expressed by the following formula.
Alanine residue content = (number of alanine residues contained in the polypeptide / number of total amino acid residues in the polypeptide) x 100 (%)
Further, the glycine residue content, the serine residue content, the threonine residue content, the proline residue content and the tyrosine residue content are as follows. It is synonymous with what is read as a threonine residue, a proline residue, and a tyrosine residue.

From the viewpoint of improving workability, it is important to inhibit strong hydrogen bonds between molecules at the time of processing, and from this viewpoint, amino acids having large side chains or flexible amino acids are homogeneous in the entire sequence to a certain extent. It is desirable that it is contained in, and specifically, a motif containing a tyrosine residue, a threonine residue, and a proline residue may be repeatedly contained in a cycle. For example, the total content of proline residue, threonine residue and tyrosine residue in any consecutive 20 amino acid residue may be 5% or more, 10% or more, or 15% or more, and 50% or less. , 40% or less, 30% or less, or 20% or less.

In the artificial structural protein according to one embodiment, the total of the serine residue content, the threonine residue content and the tyrosine residue content may be 4% or more, 4.5% or more, and 5 % Or more, 5.5% or more, 6% or more, 6.5% or more, 7% or more. The total of serine residue content, threonine residue content and tyrosine residue content may be, for example, 35% or less, 33% or less, 30% or less, 25% or less. It may be 20% or less.

The artificially structured protein according to one embodiment may have a repetitive sequence. That is, the polypeptide according to the present embodiment may have a plurality of amino acid sequences (repeated sequence units) having high sequence identity in the polypeptide. The number of amino acid residues in the repetitive sequence unit is preferably 6 to 200. The sequence identity between the repetitive sequence units may be, for example, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more. It may be 98% or more, and may be 99% or more.

The artificial structural protein according to one embodiment may _{contain (A) n} motif. In the present specification, the (A) _n motif means an amino acid sequence mainly composed of an alanine residue. (A) The _number of amino acid residues of the n motif may be 2 to 27, and may be an integer of 2 to 20, 2 to 16, or 2 to 12. Further, (A) _{the ratio of the number of alanine residues to the total number of amino acid residues in the n} motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues).

The artificial structure protein preferably has an amino acid sequence corresponding to the insertion of a cysteine residue at a position adjacent to the glycine residue, serine residue, or alanine residue, and the artificial structure protein preferably has an amino acid sequence corresponding to that of the glycine residue. It is more preferable to have an amino acid sequence corresponding to the insertion of a cysteine residue at adjacent positions. In this case, since the movable range of the side chain of the cysteine residue is wide, the mercapto group (-SH) existing in the side chain of the cysteine residue easily forms a disulfide bond within or between the molecules, which further improves the physical properties. It can be further improved. The cysteine residue may be located between the glycine residue, serine residue, or alanine residue and the glycine residue, serine residue, or alanine residue, and the serine residue and the glycine residue. It may be located between.

The artificial structure protein preferably has an amino acid sequence corresponding to the insertion of a cysteine residue at a position adjacent to the hydrophobic amino acid residue. In this case, the hydrophobic amino acid residues are fixed between the molecules by hydrophobic interaction, so that the mercapto group (-SH) at the cysteine residue easily forms a disulfide bond, further improving the physical properties. Can be done. The cysteine residue may be located next to the hydrophobic amino acid residue and may be located between the hydrophobic amino acid residue and an amino acid residue other than the hydrophobic amino acid residue, and is hydrophobic. It may be located between the amino acid residue and the glycine residue, serine residue, or alanine residue, and may be located between the hydrophobic amino acid residue and the glycine residue. The hydrophobic amino acid residue may be one selected from the group consisting of isoleucine residue, valine residue, leucine residue, phenylalanine residue, methionine residue, and alanine residue.

(Artificial fibroin)
As the artificial structural protein, artificial fibroin is preferable. Examples of fibroin include naturally occurring fibroin. Examples of naturally occurring fibroin include fibroin produced by insects or spiders.

Examples of fibroins produced by insects include Bombyx mori, Bombyx mandarina, Antheraea yamamai, Anteraea perni, tussah, and tussah. ), Silk moth (Samia synthia), Chrysanthemum (Caligra japonica), Chusser silk moth (Antheraea mylitta), Muga silk moth (Antheraea assama) Silk protein can be mentioned.

More specific examples of fibroin produced by insects include silk moth fibroin L chain (GenBank accession number M76430 (base sequence), AAA27840.1 (amino acid sequence)).

Examples of fibroins produced by spiders include spiders belonging to the genus Araneus such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders. Spiders belonging to the genus Spider, spiders belonging to the genus Pronus, spiders belonging to the genus Trinofundamashi (genus Cyrtarachne), spiders belonging to the genus Trinofundamashi, and spiders belonging to the genus Cyrtarachne. Spiders belonging to (Gasteracantha genus), spiders belonging to the genus Isekigumo (genus Ordgarius) such as Mameitaisekigumo and Mutsutogaysekigumo, spiders belonging to the genus Koganegumo, Kogatakoganegumo and Nagakoganegumo, etc. Spiders belonging to the genus Arachunura, spiders belonging to the genus Acusilas such as spiders, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora, spiders belonging to the genus Cytophora ) Spiders, spiders, spiders belonging to the genus Cyclosa, such as spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Tetragnatha, such as Yasagata spider, Harabiroashidaka spider, and Urokoa spider, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus Nephila, spiders belonging to the genus Menosira such as spiders, spiders belonging to the genus Dyschiriognatha, spiders such as spiders, spiders, spiders, spiders, spiders Spiders belonging to the genus (Latrodectus) and spiders belonging to the family Spiders (Tetragnathidae) such as spiders belonging to the genus Euprostenops. Examples include pider silk protein. Examples of the spider silk protein include traction thread proteins such as MaSp (MaSp1 and MaSp2) and ADF (ADF3 and ADF4), MiSp (MiSp1 and MiSp2), and the like.

More specific examples of fibroin produced by spiders include, for example, fibroin-3 (aff-3) [derived from Araneus diadematus] (GenBank accession numbers AAC47010 (amino acid sequence), U47855 (base sequence)), fibroin-. 4 (aff-4) [derived from Araneus diadematus] (GenBank accession number AAC47011 (amino acid sequence), U47856 (base sequence)), dragline silk protein spidroin 1 [derived from Nephila clavipes] (GenBank accession number A) U37520 (base sequence)), major angu11ate protein 1 [derived from Latrodictus hesperus] (GenBank accession number ABR68856 (amino acid sequence), EF595246 (base sequence)), dragline sirk proteinAcycla2 (Amino acid sequence), AF441245 (base sequence)), major operlate protein 1 [derived from Euprosthenops protein] (GenBank accession number CAJ00428 (amino acid sequence), AJ973155 (base sequence)), and major amplifier Protein 2 Accession number CAM32249.1 (amino acid sequence), AM490169 (base sequence)), minor amplify silk protein 1 [Nephila protein] (GenBank accession number AAC14589.1 (amino acid sequence)), minor amplifier silk protein (GenBank accession number AAC14591.1 (amino acid sequence)), minor amplify spidroin-like protein [Nephilengys cruisentata] (GenBank accession number ABR) 3727.81 (amino acid sequence) and the like can be mentioned.

As a more specific example of naturally occurring fibroin, further, fibroin whose sequence information is registered in NCBI GenBank can be mentioned. For example, among the sequence information registered in NCBI GenBank, among the sequences containing INV as DIVISION, spidroin, complete, fibroin, "silk and protein", or "silk and protein" are described as keywords in DEFINITION. It can be confirmed by extracting a sequence, a character string of a specific protein from CDS, and a sequence in which a specific character string is described in TISSUE TYPE from SOURCE.

As used herein, the term "artificial fibroin" means artificially produced fibroin (artificial fibroin). The artificial fibroin may be a fibroin having a different amino acid sequence from the naturally occurring fibroin, or may be a fibroin having the same amino acid sequence as the naturally occurring fibroin.

The artificial fibroin may be a fibrous protein having a structure similar to that of naturally occurring fibroin, and may be fibroin having a sequence similar to the repetitive sequence of naturally occurring fibroin. The "sequence similar to the repetitive sequence possessed by fibroin" may be a sequence actually possessed by naturally occurring fibroin, or may be a sequence similar thereto.

The "artificial fibroin", if it has the amino acid sequence specified in the present disclosure, is one in which the amino acid sequence is modified by relying on the naturally occurring fibroin (for example, the gene sequence of the cloned naturally occurring fibroin is modified). The amino acid sequence may be modified by the above (for example, by chemically synthesizing a nucleic acid encoding the designed amino acid sequence) by artificially designing the amino acid sequence regardless of naturally occurring fibroin. It may have a desired amino acid sequence). An artificial fibroin having a modified amino acid sequence is also included in the artificial fibroin if the amino acid sequence is different from that of the naturally occurring fibroin. Examples of artificial fibroin include artificial silk fibroin (modified amino acid sequence of silk protein produced by silkworm) and artificial spider silk fibroin (modified amino acid sequence of spider silk protein produced by spiders). ) And so on. Since artificial fibroin is relatively easy to fibrillate and has high fiber forming ability, it preferably contains artificial spider silk fibroin as a molding material, and more preferably consists of artificial spider silk fibroin.

The artificial fibroin according to the present embodiment has a domain sequence represented by the formula 1: [(A) n motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It may be a protein contained. The artificial fibroin may further have an amino acid sequence (N-terminal sequence and C-terminal sequence) added to either or both of the N-terminal side and the C-terminal side of the domain sequence. The N-terminal sequence and the C-terminal sequence are not limited to this, but are typically regions that do not have the repetition of the amino acid motif characteristic of fibroin, and consist of about 100 residues of amino acids. The chemically modified artificial fibroin does not include the introduction of a partial structure not derived from amino acids by chemical modification.

As used herein, the term "domain sequence" refers to a fibroin-specific crystalline region (typically _{corresponding to (A) n} motif of amino acid sequence) and an amorphous region (typically, REP of amino acid sequence). An amino acid sequence that produces (corresponding.)), _{Which is represented by the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _n motif. Means an array. Here, the (A) _n motif shows an amino acid sequence mainly composed of alanine residues, and the number of amino acid residues is 2-27. (A) The _number of amino acid residues of the n motif may be an integer of 2 to 20, 4 to 27, 4 to 20, 8 to 20, 10 to 20, 4 to 16, 8 to 16, or 10 to 16. .. Further, (A) _{the ratio of the number of alanine residues to the total number of amino acid residues in the n} motif may be 40% or more, 60% or more, 70% or more, 80% or more, 83% or more, 85% or more, It may be 86% or more, 90% or more, 95% or more, or 100% (meaning that it is composed of only alanine residues). A plurality of (A) _n motifs present in the domain sequence may be composed of at least 7 alanine residues only. REP shows an amino acid sequence consisting of 2 to 200 amino acid residues. REP may be an amino acid sequence composed of 10 to 200 amino acid residues. m represents an integer of 2 to 300 and may be an integer of 10 to 300. The plurality of (A) _n motifs may have the same amino acid sequence or different amino acid sequences. The plurality of REPs may have the same amino acid sequence or different amino acid sequences.

Specific examples of artificial fibroin include artificial fibroin (first artificial fibroin) derived from the large spitting tube bookmark thread protein produced in the large bottle-shaped gland of spiders, and a domain sequence with a reduced content of glycine residues. artificial fibroin having a (second artificial fibroin), (a) the artificial fibroin content of _n motifs has a domain sequences with reduced (third artificial fibroin), the content of glycine residues, and (a) _n Artificial fibroin with reduced motif content (fourth artificial fibroin), artificial fibroin with a domain sequence that locally contains a region with a high hydrophobicity index (fifth artificial fibroin), and content of glutamine residues. An artificial fibroin having a reduced domain sequence (sixth artificial fibroin) can be mentioned.

Examples of the first artificial fibroin include proteins containing a domain sequence represented by the formula 1: [(A) _n motif-REP] _m. In the first artificial fibroin, the _{number of amino acid residues of (A) n} motif is preferably an integer of 3 to 20, more preferably an integer of 4 to 20, further preferably an integer of 8 to 20, and an integer of 10 to 20. Is even more preferable, an integer of 4 to 16 is even more preferable, an integer of 8 to 16 is particularly preferable, and an integer of 10 to 16 is most preferable. In the first artificial fibroin, the number of amino acid residues constituting REP in the formula 1 is preferably 10 to 200 residues, more preferably 10 to 150 residues, and 20 to 100 residues. Is even more preferable, and 20 to 75 residues are even more preferable. In the first artificial fibroin, the total number of residues of glycine residue, serine residue and alanine residue contained in the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _{m is the amino acid residue.} It is preferably 40% or more, more preferably 60% or more, and further preferably 70% or more with respect to the total number.

The first artificial fibroin contains the unit of the amino acid sequence represented by the formula 1: [(A) _n motif-REP] _m , and the C-terminal sequence is the amino acid sequence shown in any of SEQ ID NOs: 1 to 3 or It may be a polypeptide having an amino acid sequence having 90% or more homology with the amino acid sequence shown in any of SEQ ID NOs: 1 to 3.

The amino acid sequence shown in SEQ ID NO: 1 is the same as the amino acid sequence consisting of 50 residues at the C-terminal of the amino acid sequence of ADF3 (GI: 1263287, NCBI), and the amino acid sequence shown in SEQ ID NO: 2 is a sequence. It is the same as the amino acid sequence in which 20 residues were removed from the C end of the amino acid sequence shown in No. 1, and the amino acid sequence shown in SEQ ID NO: 3 was obtained by removing 29 residues from the C end of the amino acid sequence shown in SEQ ID NO: 1. It has the same amino acid sequence.

As a more specific example of the first artificial fibroin, the amino acid sequence shown in (1-i) SEQ ID NO: 4 (recombinant spider silk protein ADF3 KaiLargeNRSH1), or the amino acid sequence shown in (1-ii) SEQ ID NO: 4 and 90 An artificial fibroin containing an amino acid sequence having a sequence identity of% or more can be mentioned. The sequence identity is preferably 95% or more.

The amino acid sequence shown in SEQ ID NO: 4 is the amino acid sequence of ADF3 having an amino acid sequence (SEQ ID NO: 5) added to the N-terminal, which consists of a starting codon, a His10 tag, and an HRV3C protease (Human rhinovirus 3C protease) recognition site. The 13th repeat region is increased approximately twice and mutated so that the translation terminates at the 1154th amino acid residue. The amino acid sequence at the C-terminal of the amino acid sequence shown in SEQ ID NO: 4 is the same as the amino acid sequence shown in SEQ ID NO: 3.

The artificial fibroin (1-i) may consist of the amino acid sequence shown in SEQ ID NO: 4.

The second artificial fibroin has an amino acid sequence whose domain sequence has a reduced content of glycine residues as compared to naturally occurring fibroin. It can be said that the second artificial fibroin has an amino acid sequence corresponding to at least one or more glycine residues in REP replaced with another amino acid residue as compared with naturally occurring fibroin. ..

The second artificial fibroin has a domain sequence of GGX and GPGXX in REP (where G is a glycine residue, P is a proline residue, and X is an amino acid residue other than glycine) as compared with naturally occurring fibroin. In at least one motif sequence selected from), it has an amino acid sequence corresponding to at least one or a plurality of glycine residues in the motif sequence being replaced with another amino acid residue. You may.

In the second artificial fibroin, the ratio of the motif sequence in which the above-mentioned glycine residue is replaced with another amino acid residue may be 10% or more with respect to the total motif sequence.

The second artificial fibroin contains the domain sequence represented by the formula 1: [(A) _n motif-REP] _m , and the domain sequence from the (A) _n motif located closest to the C-terminal side from the above domain sequence. The total number of amino acid residues in the amino acid sequence consisting of XGX (where X indicates amino acid residues other than glycine) contained in all REPs in the sequence excluding the sequence up to the C-terminal of is z, and the above domain sequence. When the total number of amino acid residues in the sequence excluding the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the above domain sequence is w, z / w is 30% or more. It may have an amino acid sequence of 40% or more, 50% or more, or 50.9% or more. (A) The _{number of alanine residues with respect to the total number of amino acid residues in the n} motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The second artificial fibroin is preferably one in which the content ratio of the amino acid sequence consisting of XGX is increased by substituting one glycine residue of the GGX motif with another amino acid residue. In the second artificial fibroin, the content ratio of the amino acid sequence consisting of GGX in the domain sequence is preferably 30% or less, more preferably 20% or less, further preferably 10% or less, 6 % Or less is even more preferable, 4% or less is even more preferable, and 2% or less is particularly preferable. The content ratio of the amino acid sequence consisting of GGX in the domain sequence can be calculated by the same method as the method for calculating the content ratio (z / w) of the amino acid sequence consisting of XGX below.

The method of calculating z / w will be described in more detail. First, in the fibroin (artificial fibroin or naturally-derived fibroin) containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m, it is located most on the C-terminal side from the domain sequence (A) _n. The amino acid sequence consisting of XGX is extracted from all REPs contained in the sequence excluding the sequence from the motif to the C end of the domain sequence. The total number of amino acid residues constituting XGX is z. For example, when 50 amino acid sequences consisting of XGX are extracted (no duplication), z is 50 × 3 = 150. Further, for example, when X (center X) contained in two XGX exists as in the case of an amino acid sequence consisting of XGXGX, the calculation is performed by deducting the overlap (in the case of XGXGX, 5 amino acid residues are used). be). w is the total number of amino acid residues contained in the sequence excluding the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. For example, in the case of the domain sequence shown in FIG. 1, w is 4 + 50 + 4 + 100 + 4 + 10 + 4 + 20 + 4 + 30 = 230 ( _{excluding the (A) n} motif located most on the C-terminal side). Next, z / w (%) can be calculated by dividing z by w.

Here, z / w in naturally derived fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types were derived from spiders) were extracted. Naturally-derived fibroin containing the domain sequence represented by the formula 1: [(A) _n motif-REP] _m among all the extracted fibroins and having an amino acid sequence consisting of GGX in the fibroin of 6% or less. From the amino acid sequence of fibroin, z / w was calculated by the above-mentioned calculation method. The result is shown in FIG. The horizontal axis of FIG. 2 indicates z / w (%), and the vertical axis indicates frequency. As is clear from FIG. 2, the z / w in naturally-derived fibroin is less than 50.9% (the highest is 50.86%).

In the second artificial fibroin, z / w is preferably 50.9% or more, more preferably 56.1% or more, further preferably 58.7% or more, and 70% or more. Is even more preferable, and 80% or more is even more preferable. The upper limit of z / w is not particularly limited, but may be, for example, 95% or less.

The second artificial fibroin is, for example, modified from the cloned naturally occurring fibroin gene sequence by substituting at least a part of the base sequence encoding the glycine residue to encode another amino acid residue. Obtainable. At this time, one glycine residue in the GGX motif and the GPGXX motif may be selected as the glycine residue to be modified, or may be replaced so that z / w is 50.9% or more. It can also be obtained, for example, by designing an amino acid sequence satisfying the above embodiment from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, in addition to the modification corresponding to the substitution of the glycine residue in REP with another amino acid residue from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted or deleted. , Insertion and / or modification of the amino acid sequence corresponding to the addition may be performed.

The other amino acid residue described above is not particularly limited as long as it is an amino acid residue other than the glycine residue, but is a valine (V) residue, a leucine (L) residue, an isoleucine (I) residue, and methionine ( Hydrophobic amino acid residues such as M) residue, proline (P) residue, phenylalanine (F) residue and tryptophan (W) residue, glutamine (Q) residue, asparagine (N) residue, serine (S) ) Residues, hydrophilic amino acid residues such as lysine (K) residue and glutamate (E) residue are preferred, valine (V) residue, leucine (L) residue, isoleucine (I) residue, phenylalanine ( F) residues and glutamine (Q) residues are more preferred, and glutamine (Q) residues are even more preferred.

As a more specific example of the second artificial fibroin, (2-i) SEQ ID NO: 6 (Met-PRT380), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met) -Contains an amino acid sequence represented by PRT799) or (2-ii) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. Artificial fibroin can be mentioned.

The artificial fibroin of (2-i) will be described. The amino acid sequence shown in SEQ ID NO: 6 is obtained by substituting GQX for all GGX in the REP of the amino acid sequence shown in SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin. _{In the amino acid sequence shown by SEQ ID NO: 7, every two (A) n} motifs are deleted from the N-terminal side to the C-terminal side from the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence is further before the C-terminal sequence. One [(A) _n motif-REP] is inserted in. In the amino acid sequence shown in SEQ ID NO: 8, two alanine residues are inserted on the C-terminal side _{of each (A) n motif of the amino acid sequence shown in SEQ ID NO: 7, and some glutamine (Q) residues are further added.} It is substituted with a serine (S) residue and a part of the amino acid on the C-terminal side is deleted so as to have substantially the same molecular weight as that of SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 9 is a region of 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (however, several amino acid residues on the C-terminal side of the region are substituted). A predetermined hinge sequence and His tag sequence are added to the C-terminal of the sequence obtained by repeating the above four times.

The value of z / w in the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) is 46.8%. The z / w values in the amino acid sequence shown in SEQ ID NO: 6, the amino acid sequence shown in SEQ ID NO: 7, the amino acid sequence shown in SEQ ID NO: 8, and the amino acid sequence shown in SEQ ID NO: 9 are 58.7%, respectively. It is 70.1%, 66.1% and 70.0%. Further, the values of x / y in the jagged ratio (described later) of 1: 1.8 to 11.3 of the amino acid sequences shown by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are They are 15.0%, 15.0%, 93.4%, 92.7% and 89.8%, respectively.

The artificial fibroin of (2-i) may consist of the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The artificial fibroin of (2-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The artificial fibroin of (2-ii) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The artificial fibroin (2-ii) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of () is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

The second artificial fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization, and the like of artificial fibroin.

Examples of the tag sequence include affinity tags that utilize specific affinity (binding, affinity) with other molecules. As a specific example of the affinity tag, a histidine tag (His tag) can be mentioned. The His tag is a short peptide in which about 4 to 10 histidine residues are lined up, and has the property of specifically binding to metal ions such as nickel. Therefore, isolation of artificial fibroin by metal chelating chromatography (chromatography). Can be used for. Specific examples of the tag sequence include the amino acid sequence shown in SEQ ID NO: 11 (amino acid sequence including His tag sequence and hinge sequence).

In addition, tag sequences such as glutathione-S-transferase (GST) that specifically binds to glutathione and maltose-binding protein (MBP) that specifically binds to maltose can also be used.

Furthermore, an "epitope tag" utilizing an antigen-antibody reaction can also be used. By adding an antigenic peptide (epitope) as a tag sequence, an antibody against the epitope can be bound. Examples of the epitope tag include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like. By utilizing the epitope tag, artificial fibroin can be easily purified with high specificity.

Further, a tag sequence in which the tag sequence can be separated by a specific protease can also be used. By treating the protein adsorbed via the tag sequence with protease, artificial fibroin from which the tag sequence has been separated can also be recovered.

As a more specific example of the artificial fibroin containing the tag sequence, the amino acids represented by (2-iii) SEQ ID NO: 12 (PRT380), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799). Examples thereof include artificial fibroins comprising a sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (2-iv) SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. ..

The amino acid sequences represented by SEQ ID NO: 16 (PRT313), SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are represented by SEQ ID NO: 10, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown by SEQ ID NO: 11 (including His tag sequence and hinge sequence) is added to the N-terminal of the indicated amino acid sequence.

The artificial fibroin of (2-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The artificial fibroin (2-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The artificial fibroin of (2-iv) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The artificial fibroin (2-iv) has 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 and is contained in REP. However, X indicates an amino acid residue other than glycine.) When the total number of amino acid residues in the amino acid sequence consisting of () is z and the total number of amino acid residues in REP in the above domain sequence is w, z / w Is preferably 50.9% or more.

The second artificial fibroin may contain a secretory signal for releasing the protein produced in the artificial protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The third artificial fibroin has an amino acid sequence _{whose domain sequence has a reduced content of (A) n motif as compared with naturally occurring fibroin.} It can be said that the domain sequence of the third artificial fibroin has an amino acid sequence corresponding to the deletion of _{at least one or more (A) n motifs as compared with naturally occurring fibroin.}

The third artificial fibroin may have an amino acid sequence corresponding to a 10-40% deletion of the _{(A) n motif from naturally occurring fibroin.}

A third artificial fibroin its domain sequence, compared to the naturally occurring fibroin, at least from the N-terminal C-terminal one to three toward the side of the (A) _n motif every one (A) _n motif It may have an amino acid sequence corresponding to the deletion of.

_{The third artificial fibroin has a domain sequence of at least two consecutive (A) n-} motif deletions and one (A) from the N-terminal side to the C-terminal side as compared to naturally occurring fibroin. ) It may have an amino acid sequence corresponding to the deletion of the _{n-motif being repeated in this order.}

The third artificial fibroin may have an amino acid sequence corresponding to the deletion _{of the (A) n} motif at least every other two domain sequences from the N-terminal side to the C-terminal side. ..

The third artificial fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _{m , and two adjacent [(A) n} motifs from the N-terminal side to the C-terminal side. -REP] When the number of amino acid residues in the REP of the unit is sequentially compared and the number of amino acid residues in the REP having a small number of amino acid residues is 1, the ratio of the number of amino acid residues in the other REP is 1.8 to 1. When x is the maximum value of the sum of the number of amino acid residues of two adjacent [(A) _{n motif-REP] units, which is 11.3, and y is the total number of amino acid residues in the domain sequence.} In addition, it may have an amino acid sequence in which x / y is 20% or more, 30% or more, 40% or more, or 50% or more. (A) The _{number of alanine residues with respect to the total number of amino acid residues in the n} motif may be 83% or more, preferably 86% or more, more preferably 90% or more, and 95% or more. It is even more preferably 100% (meaning that it is composed only of alanine residues).

The calculation method of x / y will be described in more detail with reference to FIG. FIG. 1 shows a domain sequence obtained by removing the N-terminal sequence and the C-terminal sequence from artificial fibroin. From the N-terminal side (left side), the domain sequence consists of (A) _n motif-first REP (50 amino acid residues)-(A) _n motif-second REP (100 amino acid residues)-(A) _n. Motif-Third REP (10 amino acid residues)-(A) _n Motif-Fourth REP (20 amino acid residues)-(A) _n Motif-Fifth REP (30 amino acid residues)-(A) _It has an arrangement called n motifs.

Two adjacent [(A) _n motif-REP] units are sequentially selected from the N-terminal side toward the C-terminal side so as not to overlap. At this time, _{there may be a [(A) n} motif-REP] unit that is not selected. In FIG. 1, pattern 1 (comparison between the first REP and the second REP and comparison between the third REP and the fourth REP), pattern 2 (comparison between the first REP and the second REP, and a comparison). 4th REP and 5th REP comparison), Pattern 3 (2nd REP and 3rd REP comparison, and 4th REP and 5th REP comparison), Pattern 4 (1st REP and (Comparison of the second REP) is shown. There are other selection methods.

Next, for each pattern, the number of amino acid residues of each REP in _{two adjacent [(A) n motif-REP] units selected is compared.} The comparison is performed by obtaining the ratio of the number of amino acid residues of the other when the one with the smaller number of amino acid residues is set to 1. For example, in the case of comparing the first REP (50 amino acid residues) and the second REP (100 amino acid residues), when the first REP having a smaller number of amino acid residues is 1, the second REP The ratio of the number of amino acid residues is 100/50 = 2. Similarly, in the case of comparing the 4th REP (20 amino acid residues) and the 5th REP (30 amino acid residues), when the 4th REP having a smaller number of amino acid residues is set to 1, the 5th REP The ratio of the number of amino acid residues in is 30/20 = 1.5.

In FIG. 1, when the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is 1.8 to 11.3 [(A) _n motif-REP] unit set. Shown by solid line. In the present specification, this ratio is referred to as a jagged ratio. When the one with the smaller number of amino acid residues is 1, the ratio of the number of amino acid residues of the other is less than 1.8 or more than 11.3 _{. The set of [(A) n} motif-REP] units is indicated by a broken line. Indicated.

In each pattern, add up the total number of amino acid residues of the two adjacent [(A) _n motif-REP] units shown by the solid line (not only REP, but also the number of amino acid residues of _{(A) n motif.} be.). Then, the total values added are compared, and the total value (maximum value of the total value) of the pattern in which the total value is maximized is defined as x. In the example shown in FIG. 1, the total value of pattern 1 is the maximum.

Next, x / y (%) can be calculated by dividing x by the total number of amino acid residues y in the domain sequence.

In the third artificial fibroin, x / y is preferably 50% or more, more preferably 60% or more, further preferably 65% or more, still more preferably 70% or more. It is preferably 75% or more, even more preferably 80% or more, and particularly preferably 80% or more. The upper limit of x / y is not particularly limited and may be, for example, 100% or less. When the jagged ratio is 1: 1.9 to 11.3, x / y is preferably 89.6% or more, and when the jagged ratio is 1: 1.8 to 3.4, x. / Y is preferably 77.1% or more, and when the jagged ratio is 1: 1.9 to 8.4, x / y is preferably 75.9% or more, and the jagged ratio is 1. In the case of 1.9 to 4.1, x / y is preferably 64.2% or more.

When the third artificial fibroin is an artificial fibroin in which _{at least 7 of the (A) n} motifs present in the domain sequence are composed only of alanine residues, x / y is 46.4% or more. Is more preferable, 50% or more is more preferable, 55% or more is further preferable, 60% or more is further more preferable, 70% or more is even more preferable, and 80% or more. It is particularly preferable to have. The upper limit of x / y is not particularly limited and may be 100% or less.

Here, x / y in naturally derived fibroin will be described. First, as described above, when the fibroin whose amino acid sequence information was registered in NCBI GenBank was confirmed by the method exemplified, 663 types of fibroin (of which 415 types were derived from spiders) were extracted. Of all the extracted fibroin, x / y from the amino acid sequence of naturally occurring fibroin composed of the domain sequence represented by the formula 1: [(A) _n motif-REP] _{m by the above calculation method.} Was calculated. The results when the jagged ratio is 1: 1.9 to 4.1 are shown in FIG.

The horizontal axis of FIG. 3 indicates x / y (%), and the vertical axis indicates frequency. As is clear from FIG. 3, the x / y of naturally occurring fibroin is less than 64.2% (the highest is 64.14%).

The third artificial fibroin, for example, deletes one or more of the sequences encoding the _{(A) n} motif from the cloned naturally occurring fibroin gene sequence so that x / y is 64.2% or more. Can be obtained by Further, for example, an amino acid sequence corresponding to the deletion _{of one or more (A) n} motifs so that x / y is 64.2% or more is designed and designed from the amino acid sequence of naturally occurring fibroin. It can also be obtained by chemically synthesizing a nucleic acid encoding the amino acid sequence. _{In each case, in addition to the modification corresponding to the deletion of (A) n} motif from the amino acid sequence of naturally occurring fibroin, one or more amino acid residues are further substituted, deleted, inserted and / or added. The amino acid sequence corresponding to the above may be modified.

As a more specific example of the third artificial fibroin, (3-i) SEQ ID NO: 17 (Met-PRT399), SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525) or SEQ ID NO: 9 (Met) -Contains an amino acid sequence represented by PRT799) or an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by (3-ii) SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. Artificial fibroin can be mentioned.

The artificial fibroin of (3-i) will be described. The amino acid sequence shown by SEQ ID NO: 17 is from the amino acid sequence shown by SEQ ID NO: 10 (Met-PRT313) corresponding to naturally occurring fibroin, every other (A) _{n from the N-terminal side to the C-terminal side.} The motif is deleted, and one [(A) _n motif-REP] is inserted before the C-terminal sequence. The amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 is as described in the second artificial fibroin.

The x / y value of the amino acid sequence shown in SEQ ID NO: 10 (corresponding to naturally occurring fibroin) at a jagged ratio of 1: 1.8 to 11.3 is 15.0%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 17 and the amino acid sequence shown in SEQ ID NO: 7 is 93.4%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 8 is 92.7%. The value of x / y in the amino acid sequence shown in SEQ ID NO: 9 is 89.8%. The values of z / w in the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 are 46.8%, 56.2%, 70.1% and 66. 1% and 70.0%.

The artificial fibroin of (3-i) may consist of the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

The artificial fibroin of (3-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9. The artificial fibroin of (3-ii) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The artificial fibroin (3-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _{The amino acid residue of two adjacent [(A) n} motif-REP] units having a ratio of the number of amino acid residues of REP of 1.8 to 11.3 (giza ratio of 1: 1.8 to 11.3) When x is the maximum value of the total value obtained by adding the numbers of groups and y is the total number of amino acid residues in the domain sequence, x / y is preferably 64.2% or more.

The third artificial fibroin may contain the tag sequence described above at either or both of the N-terminus and the C-terminus.

As a more specific example of the artificial fibroin containing the tag sequence, the amino acids represented by (3-iii) SEQ ID NO: 18 (PRT399), SEQ ID NO: 13 (PRT410), SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799). Examples include artificial fibroins comprising a sequence or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in (3-iv) SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. ..

The amino acid sequences shown in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 are the N-terminals of the amino acid sequences represented by SEQ ID NO: 17, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amino acid sequence shown by (including His tag sequence and hinge sequence) is added.

The artificial fibroin of (3-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

The artificial fibroin (3-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15. The artificial fibroin of (3-iv) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The artificial fibroin (3-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 18, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, and is N-terminal to C-terminal. When the number of amino acid residues of REP of two adjacent [(A) _n motif-REP] units is sequentially compared and the number of amino acid residues of REP having a small number of amino acid residues is 1, the other _Let x be the maximum value of the total value of the sum of the number of amino acid residues of two adjacent [(A) n motif-REP] units in which the ratio of the number of amino acid residues in REP is 1.8 to 11.3. , When the total number of amino acid residues in the domain sequence is y, x / y is preferably 64.2% or more.

The third artificial fibroin may contain a secretory signal for releasing the protein produced in the artificial protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The fourth artificial fibroin has an amino acid sequence whose domain sequence has a _{reduced content of (A) n} motifs and a reduced content of glycine residues as compared with naturally occurring fibroin. Have. The domain sequence of the fourth artificial fibroin lacked at least one or more (A) _n motifs as compared to naturally occurring fibroin, plus at least one or more glycine residues in the REP. It can be said that it has an amino acid sequence corresponding to being substituted with another amino acid residue. That is, the fourth artificial fibroin is an artificial fibroin having the characteristics of the above-mentioned second artificial fibroin and the third artificial fibroin. Specific aspects and the like are as described in the second artificial fibroin and the third artificial fibroin.

As more specific examples of the fourth artificial fibroin, (4-i) SEQ ID NO: 7 (Met-PRT410), SEQ ID NO: 8 (Met-PRT525), SEQ ID NO: 9 (Met-PRT799), SEQ ID NO: 13 (PRT410) ), The amino acid sequence represented by SEQ ID NO: 14 (PRT525) or SEQ ID NO: 15 (PRT799), or (4-ii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 Examples thereof include artificial fibroins containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by. Specific embodiments of the artificial fibroin comprising the amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15 are as described above.

The fifth artificial fibroin had one or more amino acid residues in the REP replaced with amino acid residues having a higher hydrophobicity index than the naturally occurring fibroin in its domain sequence, and / or REP. It may have an amino acid sequence containing a region having a large hydrophobic index locally, which corresponds to the insertion of one or a plurality of amino acid residues having a large hydrophobic index.

The region having a locally large hydrophobicity index is preferably composed of consecutive 2 to 4 amino acid residues.

The amino acid residue having a large hydrophobicity index is an amino acid selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). It is more preferably a residue.

In the fifth artificial fibroin, one or more amino acid residues in REP were replaced with amino acid residues having a higher hydrophobicity index as compared with naturally occurring fibroin, and / or one or more amino acid residues in REP. In addition to the modification corresponding to the insertion of an amino acid residue with a high hydrophobicity index, one or more amino acid residues were substituted, deleted, inserted and / or added as compared with naturally occurring fibroin. There may be a modification of the amino acid sequence corresponding to the above.

The fifth artificial fibroin, for example, leaves one or more hydrophilic amino acid residues (for example, amino acid residues having a negative hydrophobicity index) in the REP from the cloned naturally occurring fibroin gene sequence. It can be obtained by substituting for a group (eg, an amino acid residue with a positive hydrophobicity index) and / or inserting one or more hydrophobic amino acid residues in the REP. Also, for example, one or more hydrophilic amino acid residues in the REP have been replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acid residues in the REP. It can also be obtained by designing an amino acid sequence corresponding to the insertion of and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more hydrophilic amino acid residues in the REP were replaced with hydrophobic amino acid residues from the amino acid sequence of naturally occurring fibroin, and / or one or more hydrophobic amino acids in the REP. In addition to the modification corresponding to the insertion of the residue, the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be further modified.

The fifth artificial fibroin contains a domain sequence represented by the formula 1: [(A) _n motif-REP] _{m , and extends from the (A) n} motif located closest to the C-terminal side to the C-terminal of the above domain sequence. In all REPs contained in the sequence excluding the sequence from the above domain sequence, the total number of amino acid residues contained in the region where the average value of the hydrophobicity index of consecutive 4 amino acid residues is 2.6 or more is defined as p. _{When the total number of amino acid residues contained in the sequence obtained by excluding the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence is q, p / q is 6 It may have an amino acid sequence of .2% or more.

For the hydrophobicity index of amino acid residues, a known index (Hydropathy index: Kyte J, & Doolittle R (1982) "A single method for dispensing the hydropathic character of protein. 105-132) is used. Specifically, the hydrophobicity index (hydropathy index, hereinafter also referred to as “HI”) of each amino acid is as shown in Table 1 below.

The method of calculating p / q will be described in more detail. For the calculation, the sequence obtained by removing the sequence from the _{(A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence represented by the formula 1: [(A) _n motif-REP] _m. (Hereinafter referred to as "sequence A") is used. First, in all the REPs contained in the sequence A, the average value of the hydrophobicity index of consecutive 4 amino acid residues is calculated. The average value of the hydrophobicity index is obtained by dividing the total HI of each amino acid residue contained in four consecutive amino acid residues by 4 (the number of amino acid residues). The average value of the hydrophobicity index is obtained for all consecutive 4 amino acid residues (each amino acid residue is used to calculate the average value 1 to 4 times). Next, a region in which the average value of the hydrophobicity index of consecutive four amino acid residues is 2.6 or more is specified. Even if a certain amino acid residue corresponds to a plurality of "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index", it should be included as one amino acid residue in the region. become. The total number of amino acid residues contained in the region is p. Further, the total number of amino acid residues contained in the sequence A is q.

For example, when 20 consecutive "4 consecutive amino acid residues having an average value of the hydrophobicity index of 2.6 or more" are extracted (no duplication), the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2. The region of .6 or more contains 20 consecutive 4 amino acid residues (no duplication), and p is 20 × 4 = 80. Further, for example, when two "consecutive four amino acid residues having an average value of 2.6 or more of the hydrophobicity index" are duplicated by one amino acid residue, the hydrophobicity index of the consecutive four amino acid residues is present. A region having an average value of 2.6 or more contains 7 amino acid residues (p = 2 × 4-1 = 7. “-1” is a deduction for duplicates). For example, in the case of the domain sequence shown in FIG. 4, p is 7 × 4 = because there are seven “consecutive 4 amino acid residues having an average value of the hydrophobicity index of 2.6 or more” without duplication. It becomes 28. Further, for example, in the case of the domain sequence shown in FIG. 4, q is 4 + 50 + 4 + 40 + 4 + 10 + 4 + 20 + 4 + 30 = 170 (excluding the (A) _n motif existing at the end of the C-terminal side). Next, p / q (%) can be calculated by dividing p by q. In the case of FIG. 4, 28/170 = 16.47%.

In the fifth artificial fibroin, p / q is preferably 6.2% or more, more preferably 7% or more, further preferably 10% or more, and more preferably 20% or more. Even more preferably, it is even more preferably 30% or more. The upper limit of p / q is not particularly limited, but may be, for example, 45% or less.

The fifth artificial fibroin is, for example, one or more hydrophilic amino acid residues (eg, a hydrophobic index) in the REP so that the amino acid sequence of the cloned naturally occurring fibroin satisfies the above p / q condition. (Amino acid residue with a negative value) is replaced with a hydrophobic amino acid residue (for example, an amino acid residue with a positive hydrophobicity index), and / or one or more hydrophobic amino acid residues are inserted in the REP. By doing so, it can be obtained by locally modifying the amino acid sequence to include a region having a large hydrophobicity index. It can also be obtained, for example, by designing an amino acid sequence satisfying the above p / q condition from the amino acid sequence of naturally occurring fibroin and chemically synthesizing a nucleic acid encoding the designed amino acid sequence. In each case, one or more amino acid residues in the REP were replaced with amino acid residues with a higher hydrophobicity index compared to naturally occurring fibroin, and / or one or more amino acid residues in the REP. In addition to the modification corresponding to the insertion of an amino acid residue having a large hydrophobicity index, the modification corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues may be performed. ..

The amino acid residue having a large hydrophobicity index is not particularly limited, but isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A). ) Is preferable, and valine (V), leucine (L) and isoleucine (I) are more preferable.

As a more specific example of the fifth artificial fibroin, (5-i) the amino acid sequence shown by SEQ ID NO: 19 (Met-PRT720), SEQ ID NO: 20 (Met-PRT665) or SEQ ID NO: 21 (Met-PRT666), Alternatively, artificial fibroin (5-ii) containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21 can be mentioned.

The artificial fibroin of (5-i) will be described. The amino acid sequence shown in SEQ ID NO: 19 consists of 3 amino acid residues in every other REP except for the domain sequence of the terminal on the C-terminal side with respect to the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410). The amino acid sequence (VLI) is inserted at two locations, a part of the glutamine (Q) residue is replaced with a serine (S) residue, and a part of the amino acid on the C-terminal side is deleted. The amino acid sequence shown by SEQ ID NO: 20 is the amino acid sequence shown by SEQ ID NO: 8 (Met-PRT525) with one amino acid sequence (VLI) consisting of 3 amino acid residues inserted every other REP. be. The amino acid sequence shown in SEQ ID NO: 21 is the amino acid sequence shown in SEQ ID NO: 8 with two amino acid sequences (VLI) consisting of three amino acid residues inserted every other REP.

The artificial fibroin of (5-i) may consist of the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21.

The artificial fibroin of (5-ii) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21. The artificial fibroin of (5-ii) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The artificial fibroin of (5-ii) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 21, and is located most on the C-terminal side (A) _n. Amino acids contained in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p and the total number of _{amino acid residues contained in the sequence excluding the sequence from the (A) n} motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

The fifth artificial fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus.

As a more specific example of an artificial fibroin containing a tag sequence, the amino acid sequence set forth in (5-iii) SEQ ID NO: 22 (PRT720), SEQ ID NO: 23 (PRT665) or SEQ ID NO: 24 (PRT666), or (5-iv). ) Artificial fibroin comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24 can be mentioned.

The amino acid sequences shown in SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24 are the amino acid sequences shown in SEQ ID NO: 11 (His tag) at the N-terminal of the amino acid sequences shown in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively. (Including array and hinge array) is added.

The artificial fibroin of (5-iii) may consist of the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24.

The artificial fibroin of (5-iv) comprises an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24. The artificial fibroin of (5-iv) is also a protein containing a domain sequence represented by _{the formula 1: [(A) n} motif-REP] _m. The sequence identity is preferably 95% or more.

The artificial fibroin (5-iv) has 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 22, SEQ ID NO: 23 or SEQ ID NO: 24, and is located most on the C-terminal side (A) _n. Amino acids contained in the region where the average value of the hydrophobicity index of 4 consecutive amino acid residues is 2.6 or more in all REPs contained in the sequence excluding the sequence from the motif to the C-terminal of the domain sequence from the domain sequence. When the total number of residues is p and the total number of _{amino acid residues contained in the sequence excluding the sequence from the (A) n} motif located closest to the C-terminal to the C-terminal of the domain sequence from the domain sequence is q. , P / q is preferably 6.2% or more.

The fifth artificial fibroin may contain a secretory signal for releasing the protein produced in the artificial protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The sixth artificial fibroin has an amino acid sequence with a reduced content of glutamine residues as compared to naturally occurring fibroin.

The sixth artificial fibroin preferably contains at least one motif selected from the GGX motif and the GPGXX motif in the amino acid sequence of REP.

When the sixth artificial fibroin contains the GPGXX motif in the REP, the GPGXX motif content is usually 1% or more, may be 5% or more, and is preferably 10% or more. The upper limit of the GPGXX motif content is not particularly limited and may be 50% or less, or 30% or less.

In the present specification, the "GPGXX motif content" is a value calculated by the following method.

Formula 1: [(A) _n- motif-REP] _m , or Formula 2: [(A) _n- motif-REP] _m- (A) _{Fibroin containing a domain sequence represented by n-} motif (artificial fibroin or naturally derived) In (fibroin), the number of GPGXX motifs contained in the region in all REPs included in the sequence excluding the sequence from _{the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence. Let s be the number obtained by multiplying the total number by 3 (that is, corresponding to the total number of G and P in the GPGXX motif), and the _{sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence is taken from the domain sequence. The GPGXX motif content is calculated as s / t, where t is the total number of amino acid residues in all REPs excluding (A) _{n motifs.}

In the calculation of the GPGXX motif content, "the _{sequence obtained by excluding the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence from the domain sequence" is targeted at "the most C-terminal side". (A) The _{sequence from the n} motif to the C end of the domain sequence (sequence corresponding to REP) may contain a sequence having a low correlation with the sequence characteristic of fibroin, and m is small. In this case (that is, when the domain sequence is short), it affects the calculation result of the GPGXX motif content, and this effect is eliminated. When the "GPGXX motif" is located at the C-terminal of the REP, even if "XX" is, for example, "AA", it is treated as a "GPGXX motif".

FIG. 5 is a schematic diagram showing a domain sequence of artificial fibroin. A specific method for calculating the GPGXX motif content will be described with reference to FIG. First, in the domain sequence of artificial fibroin shown in FIG. 5 (“[(A) _n motif-REP] _m- (A) _n motif” type), all REPs are “located closest to the C-terminal side”. (A) Since _{it is included in the "sequence obtained by removing the sequence from the n} motif to the C-terminal of the domain sequence from the domain sequence" (the sequence shown by "region A" in FIG. 5), s is calculated. The number of GPGXX motifs is 7, and s is 7 × 3 = 21. _{Similarly, all REPs are "sequences obtained by removing the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" (the sequence shown in "Region A" in FIG. 5). Since it is contained in.), The total number t of amino acid residues in all REPs excluding the _{(A) n motif from the sequence is 50 + 40 + 10 + 20 + 30 = 150.} Next, s / t (%) can be calculated by dividing s by t, which is 21/150 = 14.0% in the case of the artificial fibroin of FIG.

The sixth artificial fibroin has a glutamine residue content of preferably 9% or less, more preferably 7% or less, further preferably 4% or less, and particularly preferably 0%. ..

In the present specification, the "glutamine residue content" is a value calculated by the following method. Formula 1: [(A) _n- motif-REP] _m , or Formula 2: [(A) _n- motif-REP] _m- (A) _{Fibroin containing a domain sequence represented by n-} motif (artificial fibroin or naturally derived) _{In fibroin), all the sequences from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In the REP, the total number of glutamine residues contained in the region is u, and the _{sequence from the (A) n} motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and (A) _n. The glutamine residue content is calculated as u / t, where t is the total number of amino acid residues in all REPs excluding the motif. In the calculation of the glutamine residue content, the _{reason why "the sequence from the (A) n} motif located on the most C-terminal side to the C-terminal of the domain sequence is excluded from the domain sequence" is the above-mentioned reason. The same is true.

The sixth artificial fibroin corresponds to its domain sequence being deleted from one or more glutamine residues in REP or replaced with other amino acid residues as compared to naturally occurring fibroin. It may have an amino acid sequence.

The "other amino acid residue" may be an amino acid residue other than the glutamine residue, but is preferably an amino acid residue having a larger hydrophobicity index than the glutamine residue. The hydrophobicity index of amino acid residues is as shown in Table 1.

As shown in Table 1, amino acid residues having a larger hydrophobicity index than glutamine residues include isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), and methionine (M). ) Amino acid residues selected from alanine (A), glycine (G), threonine (T), serine (S), tryptophan (W), tyrosine (Y), proline (P) and histidine (H). can. Among these, amino acid residues selected from isoleucine (I), valine (V), leucine (L), phenylalanine (F), cysteine (C), methionine (M) and alanine (A) are more preferable. , Isoleucine (I), valine (V), leucine (L) and phenylalanine (F) are more preferably amino acid residues.

The sixth artificial fibroin has a REP hydrophobicity of -0.8 or more, more preferably -0.7 or more, further preferably 0 or more, and 0.3 or more. Is even more preferable, and 0.4 or more is particularly preferable. The upper limit of the hydrophobicity of REP is not particularly limited and may be 1.0 or less, or 0.7 or less.

In the present specification, the "hydrophobicity of REP" is a value calculated by the following method.

Formula 1: [(A) _n- motif-REP] _m , or Formula 2: [(A) _n- motif-REP] _m- (A) _{Fibroin containing a domain sequence represented by n-} motif (artificial fibroin or naturally derived) _{In fibroin), all the sequences from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence are excluded from the domain sequence (the sequence corresponding to "region A" in FIG. 5). In the REP of, the sum of the hydrophobicity indexes of each amino acid residue in the region is v, and the _{sequence from the (A) n} motif located most on the C-terminal side to the C-terminal of the domain sequence is removed from the domain sequence, and further ( A) The degree of hydrophobicity of REP is calculated as v / t, where t is the total number of amino acid residues of all REPs excluding the _{n motif.} In the calculation of the hydrophobicity of REP, the _{reason for targeting "the sequence obtained by excluding the sequence from the (A) n} motif located closest to the C-terminal side to the C-terminal of the domain sequence from the domain sequence" is the above-mentioned reason. The same is true.

The sixth artificial fibroin had its domain sequence deleted of one or more glutamine residues in REP as compared to naturally occurring fibroin, and / or one or more glutamine residues in REP. In addition to the modification corresponding to the replacement of one or more amino acid residues with other amino acid residues, there may be further modification of the amino acid sequence corresponding to the substitution, deletion, insertion and / or addition of one or more amino acid residues. ..

The sixth artificial fibroin, for example, deletes one or more glutamine residues in REP from the cloned naturally occurring fibroin gene sequence and / or removes one or more glutamine residues in REP. It can be obtained by substituting with the amino acid residue of. Also, for example, one or more glutamine residues in REP were deleted from the amino acid sequence of naturally occurring fibroin, and / or one or more glutamine residues in REP were replaced with other amino acid residues. It can also be obtained by designing an amino acid sequence corresponding to this and chemically synthesizing a nucleic acid encoding the designed amino acid sequence.

As more specific examples of the sixth artificial fibroin, (6-i) SEQ ID NO: 25 (Met-PRT888), SEQ ID NO: 26 (Met-PRT965), SEQ ID NO: 27 (Met-PRT889), SEQ ID NO: 28 (Met) -PRT916), SEQ ID NO: 29 (Met-PRT918), SEQ ID NO: 30 (Met-PRT699), SEQ ID NO: 31 (Met-PRT698), SEQ ID NO: 32 (Met-PRT966), SEQ ID NO: 41 (Met-PRT917) or SEQ ID NO: An artificial fibroin containing the amino acid sequence represented by No. 42 (Met-PRT1028), or (6-ii) SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31. , Artificial fibroin containing an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42.

The artificial fibroin of (6-i) will be described. The amino acid sequence shown in SEQ ID NO: 25 is obtained by substituting VL for all QQs in the amino acid sequence (Met-PRT410) shown in SEQ ID NO: 7. The amino acid sequence shown in SEQ ID NO: 26 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with TS, and the remaining Qs are replaced with A. The amino acid sequence shown in SEQ ID NO: 27 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with VL, and the remaining Qs are replaced with I. The amino acid sequence shown in SEQ ID NO: 28 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with VI, and the remaining Qs are replaced with L. The amino acid sequence shown in SEQ ID NO: 29 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with VF, and the remaining Qs are replaced with I.

The amino acid sequence shown in SEQ ID NO: 30 is obtained by substituting VL for all QQs in the amino acid sequence (Met-PRT525) shown in SEQ ID NO: 8. The amino acid sequence shown in SEQ ID NO: 31 is one in which all QQs in the amino acid sequence shown in SEQ ID NO: 8 are replaced with VL, and the remaining Qs are replaced with I.

In the amino acid sequence shown by SEQ ID NO: 32, all the QQs in the sequence in which the regions of the 20 domain sequences existing in the amino acid sequence shown in SEQ ID NO: 7 (Met-PRT410) are repeated twice are replaced with VF. And the remaining Q is replaced with I.

In the amino acid sequence (Met-PRT917) shown in SEQ ID NO: 41, all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with LI, and the remaining Qs are replaced with V. In the amino acid sequence (Met-PRT1028) shown in SEQ ID NO: 42, all QQs in the amino acid sequence shown in SEQ ID NO: 7 are replaced with IF, and the remaining Qs are replaced with T.

The amino acid sequences shown in SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 are all residual glutamine. The group content is 9% or less (Table 2).

The artificial fibroin of (6-i) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42. It may consist of the indicated amino acid sequence.

The artificial fibroin (6-ii) has SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 or SEQ ID NO: 42. It contains an amino acid sequence having 90% or more sequence identity with the indicated amino acid sequence. The artificial fibroin of (6-ii) is also a domain represented by _{the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It is a protein containing a sequence. The sequence identity is preferably 95% or more.

The artificial fibroin of (6-ii) preferably has a glutamine residue content of 9% or less. Further, the artificial fibroin of (6-ii) preferably has a GPGXX motif content of 10% or more.

The sixth artificial fibroin may contain a tag sequence at either or both of the N-terminus and the C-terminus. This enables isolation, immobilization, detection, visualization, and the like of artificial fibroin.

As a more specific example of the artificial fibroin containing the tag sequence, (6-iii) SEQ ID NO: 33 (PRT888), SEQ ID NO: 34 (PRT965), SEQ ID NO: 35 (PRT889), SEQ ID NO: 36 (PRT916), SEQ ID NO: 37 (PRT918), an artificial fibroin containing the amino acid sequence set forth in SEQ ID NO: 38 (PRT699), SEQ ID NO: 39 (PRT698), SEQ ID NO: 40 (PRT966), SEQ ID NO: 43 (PRT917) or SEQ ID NO: 44 (PRT1028), or ( 6-iv) Amino acid sequences and 90s shown by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44. An artificial fibroin containing an amino acid sequence having a sequence identity of% or more can be mentioned.

The amino acid sequences shown by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 are, respectively, SEQ ID NO: 25. , SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 41 and SEQ ID NO: 42 shown by SEQ ID NO: 11 at the N-terminal of the amino acid sequence. The amino acid sequence (including His tag sequence and hinge sequence) is added. Since only the tag sequence was added to the N-terminal, there was no change in the glutamine residue content, and SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39. , SEQ ID NO: 40, SEQ ID NO: 43 and SEQ ID NO: 44 all have a glutamine residue content of 9% or less (Table 3).

The artificial fibroin of (6-iii) has SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44. It may consist of the indicated amino acid sequence.

The artificial fibroin (6-iv) is represented by SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 44. It contains an amino acid sequence having 90% or more sequence identity with the indicated amino acid sequence. The artificial fibroin of (6-iv) is also a domain represented by _{the formula 1: [(A) n} motif-REP] _m or the formula 2: [(A) _n motif-REP] _m- (A) _{n motif.} It is a protein containing a sequence. The sequence identity is preferably 95% or more.

The artificial fibroin (6-iv) preferably has a glutamine residue content of 9% or less. Further, the artificial fibroin (6-iv) preferably has a GPGXX motif content of 10% or more.

The sixth artificial fibroin may contain a secretory signal for releasing the protein produced in the artificial protein production system to the outside of the host. The sequence of the secretory signal can be appropriately set according to the type of host.

The artificial fibroin is at least two or more of the characteristics of the first artificial fibroin, the second artificial fibroin, the third artificial fibroin, the fourth artificial fibroin, the fifth artificial fibroin, and the sixth artificial fibroin. It may be an artificial fibroin having the above-mentioned characteristics.

The protein used in the present invention (for example, artificial protein, artificial fibroin) is preferably a protein having a large total number of residues of cysteine, lysine, arginine, asparagine and glutamine, and further, the total number of cysteine, lysine, arginine, asparagine and glutamine. A protein having a large number of residues is preferable, a protein having a large total number of cysteine, lysine and arginine residues is preferable, a protein having a large total number of cysteine and lysine residues is preferable, and a protein having a large number of cysteine residues is preferable. Most preferred.

(Compound represented by the formulas (1a), (2a), (2b) or (3a))
The compound represented by the formula (1a) covalently bonds with artificial fibroin by chemically reacting with a hetero atom (for example, sulfur atom, nitrogen atom, oxygen atom) contained in the above-mentioned protein. Heteroatoms are present at substituents such as sulfhydryl group (-SH), guanidine group (-NHC (= NH) NH ₂ ), amide group (-CONH-, -CONH ₂ ), hydroxy group (-OH) and the like. For example, protein cross-linking involves amino acid residues (ie, cysteine (C), arginine (R), asparagine (N), glutamine (Q), serine (ie)) having the above-mentioned substituents in the side chain of the amino acid sequence. S), threonine (T), etc.) can be used as a foothold to crosslink via the compound represented by the formula (1a).

［式中、Ｌは２価の有機基を示す。］ The compound represented by the formula (1a) has the following chemical formula, and two maleimide groups are bonded by the linker L. The linker L may be a divalent organic group, for example, C _1-20 alkylene group, C _6-20 arylene group, C _6-20 arylene group-C _1-20 alkylene group, C _6-20 arylene group. -C _1-20 alkylene group-C _6-20 arylene group or C _1-20 alkylene group-C _6-20 arylene group-C _1-20 alkylene group, polyethylene glycol group, polypropylene glycol group, poly (ethylene glycol / It is a propylene glycol) group and a tetramethylene glycol group.

[In the formula, L represents a divalent organic group. ]

The compound represented by the formula (2a) or (2b) is covalently bonded to artificial fibroin by chemically reacting with a hetero atom (for example, sulfur atom, nitrogen atom, oxygen atom) contained in the above-mentioned protein. Heteroatoms are present at substituents such as sulfhydryl group (-SH), guanidine group (-NHC (= NH) NH ₂ ), amide group (-CONH-, -CONH ₂ ), hydroxy group (-OH) and the like. For example, protein cross-linking involves amino acid residues (ie, cysteine (C), arginine (R), asparagine (N), glutamine (Q), serine (ie)) having the above-mentioned substituents in the side chain of the amino acid sequence. S), threonine (T), etc.) can be used as a foothold for cross-linking via a compound represented by the formula (2a) or (2b).

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］ The compound represented by the formula (2a) or (2b) has the following chemical formula, and two maleimide groups are bonded by the linker L. The linker L may be a divalent organic group, for example, C _1-20 alkylene group, C _6-20 arylene group, C _6-20 arylene group-C _1-20 alkylene group, C _6-20 arylene group. -C _1-20 alkylene group-C _6-20 arylene group or C _1-20 alkylene group-C _6-20 arylene group-C _1-20 alkylene group, polyethylene glycol group, polypropylene glycol group, poly (ethylene glycol / It is a propylene glycol) group and a tetramethylene glycol group.

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

The compound represented by the formula (3a) covalently bonds with artificial fibroin by chemically reacting with a hetero atom (for example, sulfur atom, nitrogen atom, oxygen atom) contained in the above-mentioned protein. Heteroatoms are present at substituents such as sulfhydryl group (-SH), guanidine group (-NHC (= NH) NH ₂ ), amide group (-CONH-, -CONH ₂ ), hydroxy group (-OH) and the like. For example, protein cross-linking involves amino acid residues (ie, cysteine (C), arginine (R), asparagine (N), glutamine (Q), serine (ie)) having the above-mentioned substituents in the side chain of the amino acid sequence. S), threonine (T), etc.) can be used as a foothold to crosslink via the compound represented by the formula (3a).

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］ The compound represented by the formula (3a) has the following chemical formula, and two maleimide groups are bonded by the linker L. The linker L may be a divalent organic group, for example, C _1-20 alkylene group, C _6-20 arylene group, C _6-20 arylene group-C _1-20 alkylene group, C _6-20 arylene group. -C _1-20 alkylene group-C _6-20 arylene group or C _1-20 alkylene group-C _6-20 arylene group-C _1-20 alkylene group, polyethylene glycol group, polypropylene glycol group, poly (ethylene glycol / It is a propylene glycol) group and a tetramethylene glycol group.

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

The C _1-20 alkylene group is an alkylene group having 1 to 20 carbon atoms, and is, for example, a methylene group, an ethylene group, a propylene group, a butylene group, a pentylene group, a hexylene group, a heptylene group, an octylene group, a nonylene group and a decylene. Examples include a group, an undecylene group, and a dodecylene group.

The C _6-20 arylene group is an arylene group having 6 to 20 carbon atoms, and examples thereof include a phenylene group, a naphthylene group, a terphenylene group, a pyrenylene group, and a biphenylene group.

The C _6-20 arylene group-C _1-20 alkylene group is a divalent group in which an alkylene having 1 to 20 carbon atoms and an arylene group having 6 to 20 carbon atoms are covalently bonded. C _6-20 arylene group-C _1-20 alkylene group-C _6-20 arylene group and C _1-20 alkylene group-C _6-20 arylene group-C _1-20 alkylene group have carbon atoms, respectively. It is a divalent group in which 1 to 20 alkylenes and an arylene group having 6 to 20 carbon atoms are covalently bonded. Specific examples thereof include a methylene diphenylene group and a phenylene bis (methylene) group.

(Crosslink protein)
The crosslinked protein is obtained by reacting the protein with a compound represented by the formula (1a), (2a), (2b) or (3a). As a method for chemically modifying a protein, a method well known to those skilled in the art can be used. For example, a cysteine (sulfhydryl group) in a protein undergoes a 1,4-addition reaction with a maleimide group in a compound represented by the formulas (1a), (2a), (2b) or (3a). A linker (crosslinked portion) represented by (1), (2) or (3) can be introduced.

For example, the protein and the compound represented by the formula (1a), (2a), (2b) or (3a) may be mixed in a solvent. If the reaction is difficult to proceed, a base may be added or heated. The reaction temperature is usually 0 to 60 ° C, preferably 30 to 55 ° C or 35 to 50 ° C. The solvent used may be any solvent that can dissolve the artificial fibroin and does not inhibit the progress of the desired reaction. Examples of such a solvent include hexafluoroisopropyl alcohol (HFIP), dimethyl sulfoxide, formic acid, dimethylformamide, acetic acid, dimethylacetamide, and N-methylpyrrolidone.

To terminate the reaction, for example, ethyl acetate may be added to aggregate the product (chemically modified protein). The product can then be purified by washing the aggregated product with ethyl acetate, methanol, or the like.

After isolating the crosslinked protein obtained by the above method, it can be used for producing a protein molded product. In addition, the crosslinked protein can also be produced along with the production of the protein molded product. That is, the cross-linking reaction of the protein is carried out under the heating and pressurizing conditions when the composition containing the protein and the compound represented by the formula (1a), (2a), (2b) or (3a) is molded by the method described later. Can progress. By performing the cross-linking reaction and the molding step at the same time, a desired protein molded product containing the cross-linked protein can be obtained by a simpler method.

In the reaction, for example, the protein and the compound represented by the formula (1a), (2a), (2b) or (3a) may be mixed in a solvent. If the reaction is difficult to proceed, a base may be added or heated. The reaction temperature is usually 0 to 60 ° C, preferably 30 to 55 ° C or 35 to 50 ° C. The solvent used may be one that can dissolve the protein and does not inhibit the progress of the desired reaction. Examples of such a solvent include hexafluoroisopropyl alcohol (HFIP), dimethyl sulfoxide, formic acid, dimethylformamide, acetic acid, dimethylacetamide, and N-methylpyrrolidone.

To terminate the reaction, for example, ethyl acetate may be added to aggregate the product (chemically modified artificial fibroin). The product can then be purified by washing the aggregated product with ethyl acetate, methanol, or the like.

The protein molded product can also be used as a material for molding processing. The shape of the protein molding can be powdery. The crosslinked protein is not particularly limited, but can be molded by a method well known in the art such as extrusion molding, injection molding, film casting, spinning, and heat and pressure molding. The shape of the crosslinked protein molded product can be plate-like, particle-like, lump-like, film-like, or fibrous.

［式中、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］ [Second Embodiment]
A second embodiment of the present invention is a method for producing a molded product of a composition containing a crosslinked protein, which comprises a protein and a compound represented by the formulas (1a), (2a), (2b) or (3a). A method for producing a molded product, which comprises a step of heating the composition.

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

As the protein and the compound represented by the formula (1a), (2a), (2b) or (3a), those described in the first embodiment can be used.

When the shape of the protein molded product is fibrous, the molding process is, for example, spinning. Spinning can be carried out by a method well known to those skilled in the art, such as dry spinning, wet spinning, and dry wet spinning. In the spinning process, the protein cross-linking reaction can proceed by heating when the undried fibers are dried.

When the shape of the protein compact is film-like, the molding step is, for example, film casting. After casting and molding the protein and the composition containing the compound represented by the formula (1a), (2a), (2b) or (3a), the cross-linking reaction of the protein proceeds by heating when the undried film is dried. obtain.

When a composition containing a protein and a compound represented by the formulas (1a), (2a), (2b) or (3a) is heated and pressed, the protein and the formulas (1a), (2a), (2b) are heated and pressurized. Alternatively, not only the chemical reaction of the compound represented by (3a) proceeds to form a covalent bond and the cross-linking of the protein proceeds, but also the obtained cross-linked protein can be formed by resinification. Here, "resinification of the crosslinked protein" means that the phase changes to a phase having an apparently uniform appearance (apparently uniform phase), and may have translucency. Further, the molded product that has been resinified by heating and pressurizing here can also be said to be a heated and pressure molded product.

In the present specification, "translucency" means the property of transmitting light to the opposite side surface when light (for example, visible light) is irradiated from a certain direction to the object, and the appearance is not necessarily transparent. It does not matter whether it is opaque or opaque. The term "translucent" means that, for example, when observed from one side of a heat-press molded product, the presence of an object on the opposite side can be recognized. It is more preferable that the transmittance is extremely high and the shape of the object on the opposite side can be clearly seen (that is, transparent), but the outline of the object is blurred and cannot be clearly recognized like frosted glass (that is, transparent). It may be translucent). Further, the term "having translucency" also includes a case where the shape of an object on the opposite side looks distorted due to a difference in refractive index or the like. The translucency can be visually determined, but when an optical transmittance measuring device is used and the integration time is 0.1 seconds in the wavelength range of 220 to 800 nm, the transmittance is 40% or more. Suitable. On the other hand, "impermeable" means a property that does not transmit light (for example, visible light). The transmittance can be calculated, for example, based on the value of the brightness of the white light measured by irradiating the material according to the present invention with white light and using a spectrophotometer.

For example, in the step of heating a composition containing a protein and a compound represented by the formula (1a), (2a), (2b) or (3a), the composition is introduced into a mold and heated and pressurized. It may be molded by. After a step of heating and pressurizing a composition containing a protein and a compound represented by the formulas (1a), (2a), (2b) or (3a), a protein molded product (heated and pressure molded product) is obtained. Be done.

The protein molded product can be produced, for example, by heating and pressurizing a composition containing a protein and a compound represented by the formula (1a), (2a), (2b) or (3a) using a pressure molding machine. .. FIG. 6 is a schematic cross-sectional view of a pressure molding machine that can be used to manufacture a heat-press molded body. The pressure molding machine 10 shown in FIG. 6 includes a mold 2 having a through hole formed and capable of heating, and an upper pin 4 and a lower pin 6 capable of moving up and down in the through hole of the mold 2. Is. In the pressure molding machine 10, the protein composition is introduced into the gap formed by inserting the upper pin 4 or the lower pin 6 into the through hole of the mold 2, and the upper pin 4 is heated while heating the mold 2. By compressing the protein composition with the lower pin 6 and the lower pin 6, a protein molded product can be obtained.

FIG. 7 shows a process diagram for obtaining a protein molded product, in which (a) is before the introduction of the composition, (b) is immediately after the introduction of the composition, and (c) is heating and pressurizing the composition. It is a schematic cross-sectional view of the pressure molding machine of the state. As shown in FIG. 7A, the composition is introduced into the through hole with only the lower pin 6 inserted into the through hole of the mold 2. Subsequently, as shown in FIG. 7B, the upper pin 4 is inserted into the through hole of the mold 2 and lowered, the heating of the mold 2 is started, and the composition 8a before heating and pressurizing is formed through the through hole. Heat and pressurize inside. The upper pin 4 is lowered until a predetermined pressing force is reached, and heating and pressurization are continued until the composition reaches a predetermined temperature in the state shown in FIG. 7 (c), and the composition after heating and pressurizing. Obtain 8b. Then, the temperature of the mold 2 is lowered by using a cooler (for example, a spot cooler), and when the molding composition 8b reaches a predetermined temperature, the upper pin 4 or the lower pin 6 is removed from the mold 2 and the contents are removed. Take out things. Regarding the pressurization, the upper pin 4 may be lowered while the lower pin 6 is fixed, or the upper pin 4 may be lowered and the lower pin 6 may be raised. The removed contents are dried to obtain a molding composition. Regarding the pressurization, the upper pin 4 may be lowered while the lower pin 6 is fixed, or the upper pin 4 may be lowered and the lower pin 6 may be raised.

The heating is preferably performed at a mold temperature of 80 to 300 ° C., more preferably 100 to 180 ° C., still more preferably 100 to 130 ° C. Pressurization is preferably performed at 5 kN or higher, more preferably 10 kN or higher, and even more preferably 20 kN or higher. Further, after reaching a predetermined heating and pressurizing condition, the time (heat retention condition) for continuing the treatment under that condition is preferably 0 to 100 minutes, more preferably 1 to 50 minutes, still more preferably 5 to 30 minutes.

By changing the shape of the mold used, a protein molded product (heat-pressurized molded product) having a desired shape can be obtained. For example, a mold having a special shape may be used to form a shogi or chess piece, a dice, or the like into an uneven shape.

［式中、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］

［式中、Ｒは水素原子又は有機基を示し、Ｌは２価の有機基を示す。］ [Third Embodiment]
A third embodiment of the present invention comprises a base material layer and a skin layer bonded onto the base material layer, and the skin layer is represented by a protein represented by the formula (1), (2) or (3). It is an artificial leather containing a molded product containing a crosslinked protein crosslinked by a linker to be made.

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

Since artificial leather can be artificially mass-produced, it is used for a wide range of purposes as a surface cover for clothing, shoes, bags and other portable items, furniture, etc., instead of natural leather, which has a limited supply. There is. Animal leather has a subcutaneous tissue on the innermost side, and a collagen layer, a grain layer, and an epidermis layer (outermost surface) are formed from there to the outside. Hair grows from the grain layer. In the manufacturing process of natural leather (for example, cowhide), the epidermis layer and hair are removed by a processing process, and the subcutaneous tissue is removed by a lining process. Therefore, natural leather is formed of a collagen layer and a grain layer. The grain layer has a dense fiber structure close to that of the epidermis layer, and is used as the epidermis of natural leather.

On the other hand, artificial leather is a structure similar to natural leather. In Japan, artificial leather and synthetic leather are distinguished based on the difference in structure. The first aspect is called "artificial leather" in Japan, and is generally a non-woven fabric layer in which ultrafine fibers made of nylon or polyester are entangled and further impregnated with polyurethane resin, and a surface resin made of polyurethane on the non-woven fabric layer. Layers are formed. Such artificial leather exhibits a texture similar to that of natural leather. The second aspect is called "synthetic leather" in Japan, and is generally composed of a woven fabric or knitted fabric (base fabric layer) and a resin layer formed by thickly applying a polyurethane resin. There is.
As used herein, the term "artificial leather" means a structure including a base material layer and an epidermis layer, and is a material that is artificially manufactured and is a substitute for natural leather. In addition, the term "artificial leather" as used herein includes both "synthetic leather" and "artificial leather" as defined in Japan.

<Synthetic leather>
The artificial leather is shown in FIG. FIG. 8A is a schematic cross-sectional view of synthetic leather. The base material layer is a non-woven fabric layer 13 substantially composed of the non-woven fabric 11 and the crosslinked protein 12. The crosslinked protein 12 used in the non-woven fabric layer 13 may be the same as the crosslinked protein used in the epidermis layer 15, or may be different from each other. The non-woven fabric 11 used for the non-woven fabric layer 13 may be a cloth in which ultrafine fibers made of nylon or polyester are entangled. The non-woven fabric layer 13 can be obtained by impregnating the composition containing the crosslinked protein 12 with the non-woven fabric 11 and performing a heat-pressurizing treatment. As the epidermis layer 15, the protein molded product according to the first embodiment can be used. The skin layer 15 may be subjected to desired processing (dyeing, painting, graving, shrinking, embossing, etc.) so as to give an appearance closer to that of natural leather. If necessary, the adhesive layer 14 may be provided between the base material layer and the epidermis layer by a method well known to those skilled in the art.

The synthetic leather according to the present embodiment includes a base material layer containing a non-woven fabric and a polymer. The base material layer can be a layer in which the non-woven fabric and an impregnated body made of a polymer impregnated in the non-woven fabric are integrated, or a layer in which the non-woven fabric is embedded in a layer formed of the polymer. can.

In this embodiment, at least one of the non-woven fabric and the polymer contains a protein. That is, only the non-woven fabric may contain protein (polymer does not contain protein), only the polymer may contain protein (non-woven fabric does not contain protein), and both the non-woven fabric and the polymer contain protein. May be. Further, it is preferable that at least the non-woven fabric as the main material contains a protein.

(Non-woven fabric)
The non-woven fabric preferably contains a protein from the viewpoint of hygroscopicity and strength. The protein may be a structural protein, preferably artificial fibroin, and more preferably artificial spider silk fibroin. The protein may be contained in the non-woven fabric as a protein fiber. Preferred embodiments of the protein will be described later.

The non-woven fabric can be produced by a known production method, for example, using fibers containing at least a part of protein fibers. Specifically, for example, a web (including a single-layer web and a laminated web) is formed from fibers containing at least a part of protein fibers by a dry method, a wet method, an air-laid method, or the like, and then a chemical bond method is used. A non-woven fabric can be obtained by bonding the fibers of the web by (immersion method, spray method, etc.) and needle punching method.

Non-woven fabrics also include, for example, the protein in a solvent such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, or hexafluoroisopropanol (HFIP), optionally as an inorganic as a dissolution accelerator. It can also be obtained by adding it together with a salt and dissolving it to prepare a dope solution, and then spinning the dope solution by an electrospinning method (electrostatic spinning method).

The non-woven fabric is appropriately set so that the numerical ranges such as fiber density (weight), porosity, bulk density, etc. are within a range in which, for example, waterproofness and moisture permeability can be sufficiently ensured. The basis weight, porosity, bulk density, etc. can be adjusted by, for example, increasing or decreasing the amount of fibers constituting the web, or in the case of a laminated web, increasing or decreasing the number of layers.

(polymer)
The polymer is generally impregnated in a non-woven fabric in a solution state, solidified (solidified) by desolvation, and exists in a state of being fixed to the fibers in the gaps between the fibers of the non-woven fabric. That is, the polymer constitutes a base material layer of synthetic leather together with the non-woven fabric as a so-called impregnated body integrated with the non-woven fabric, and the fibers of the non-woven fabric are connected to each other to maintain the shape of the base material layer. A predetermined strength is imparted to the base material layer. Examples of such polymer substances include synthetic resins such as polyvinyl chloride, polyolefin, polystyrene, polyurethane, polyester resin, epoxy resin, acrylic polymer, and acrylonitrile polymer, and proteins. Examples of the polyurethane include polyether polyurethane, polyester polyurethane, polycarbonate polyurethane and the like, and these may be used alone or in combination of two or more. Polyurethane can be synthesized, for example, by reacting a diisocyanate with a polyol to synthesize a prepolymer, and then reacting the prepolymer with a chain extender. The diisocyanate may be an aromatic compound or an aliphatic compound. The polyol may be, for example, polyester, polyether, polycarbonate, silicon, fluororesin or the like. When a polyether is used as the polyol, it is excellent in hydrolysis resistance, mold resistance and cold resistance, and when a polycarbonate is used as the polyol, it is excellent in hydrolysis resistance and mold resistance. The chain extender may be, for example, glycol, diamine, reaction terminator or the like. The polymer preferably contains a protein from the viewpoint of hygroscopicity and strength. The protein may be a structural protein, preferably artificial fibroin, and more preferably artificial spider silk fibroin. Preferred embodiments of the protein will be described later.

The base material layer may contain a braided body in addition to the non-woven fabric and the polymer. The knitted body is a general term for knitted fabrics and woven fabrics. The knitted fabric is a knitted fabric having a weft knitting structure such as a horizontal knitting or a circular knitting (also simply referred to as a "weft knitting fabric"), or a knitted fabric having a warp knitting structure such as tricot or Russell (also simply referred to as a "warp knitted fabric"). It may be any of.). The woven fabric may be a woven fabric having any of a plain weave, a twill weave, and a satin weave. The knitted or woven body may be an unprocessed knitted or woven body obtained by knitting or weaving, or may be a knitted or woven body that has been subjected to a process such as water repellent treatment after knitting or weaving.

The braided body can be obtained by knitting or weaving a raw material yarn. A known method can be used as the knitting method and the weaving method. As the knitting machine to be used, for example, a circular knitting machine, a warp knitting machine, a flat knitting machine and the like can be used, and from the viewpoint of productivity, the use of a circular knitting machine is preferable. Examples of the flat knitting machine include a molding knitting machine and a non-sewn knitting machine, but it is more preferable to use a non-sewn knitting machine because the knitted fabric can be manufactured in the form of a final product. Examples of the loom used include a shuttle loom and a non-shuttle loom such as a gripper loom, a rapier loom, and an air jet loom.

The raw material yarn may be a single yarn, a composite yarn (for example, a blended yarn, a mixed fiber yarn, a covering yarn, etc.), or a combination thereof may be used. The single yarn and the composite yarn may be spun yarn in which short fibers are twisted, or may be filament yarn in which long fibers are twisted. Examples of the fiber contained in the raw material yarn include protein fiber, synthetic fiber such as nylon, polyester and polytetrafluoroethylene, regenerated fiber such as cupra, rayon and lyocell, and natural fiber such as cotton, hemp and silk.

When the base material layer contains a braided body in addition to the non-woven fabric and the polymer, the knitted body may be included in the base material layer as, for example, a non-woven fabric bonded to both sides thereof and integrated.

The thickness of the base material layer is not particularly limited, but may be, for example, 100 to 2000 μm, 100 to 1000 μm, 500 to 1000 μm, or 500 to 750 μm.

(Manufacturing method of synthetic leather)
In the method for producing synthetic leather according to the present embodiment, for example, a step of impregnating a non-woven fabric with an impregnating solution containing a polymer substance and then coagulating the polymer substance to form a base material layer (base material layer forming step) is performed. Be prepared. The non-woven fabric, the polymer substance and the base material layer can be applied in the same manner as those described above.

In the base material layer forming step, for example, the non-woven fabric is immersed in the impregnating liquid or the non-woven fabric is sprayed with the impregnating liquid to bring the non-woven fabric and the impregnating liquid into contact with each other to impregnate the non-woven fabric with the impregnating liquid and then solidify. It can be carried out by a method (wet method) or the like in which the components in the impregnating liquid are solidified by contacting (for example, dipping) the liquid to form a base material layer. Further, depending on the type of solvent (for example, one having a low boiling point), the non-woven fabric impregnated with the impregnating liquid as described above is desolvated by volatilizing the solvent of the impregnating liquid without contacting the coagulating liquid. It is also possible to carry out the base material layer forming step by a method of solidifying the components in the impregnating liquid to form the base material layer or the like.

The solvent used for the impregnating solution can be appropriately selected depending on the type of the polymer substance, for example, dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, alcohol or hexafluoroisopropanol (HFIP) and the like. Organic solvent of.

FIG. 12 is a schematic view showing an example of a manufacturing apparatus for manufacturing synthetic leather by a wet method. In the manufacturing method using the manufacturing apparatus 800 shown in FIG. 12, first, the non-woven fabric 81 is pulled out from the roll around which the non-woven fabric 81 is wound, and the non-woven fabric 81 is immersed in the impregnation tank 82 containing the impregnating liquid. Next, the polymer substance impregnated in the non-woven fabric 81 is coagulated in the coagulation tank 83 containing the coagulation liquid. Then, after washing through a hot water washing tank 84 containing a washing liquid, the synthetic leather is dried in a dryer 85, and the synthetic leather 86 is wound on a roll to produce the synthetic leather.

The type of coagulating liquid can be appropriately selected depending on the types of the non-woven fabric and the polymer substance, and the type of the solvent of the impregnating liquid. For example, when the polymer substance contains polyurethane, for example, N, N-dimethylformamide can be used as the solvent for the impregnating liquid, and water (hot water) can be used as the coagulating liquid. Further, for example, when the polymer substance contains a protein, for example, dimethylsulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP) as a solvent for the impregnating solution, and dissolved in these. A product to which an inorganic salt is added as an accelerator, lower alcohol having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, acetone, and water (hot water) can be used as the coagulation liquid. Various additives may be added to the coagulation liquid as needed.

The manufacturing method according to the present embodiment may further include a finishing step (surface grinding, coating, dyeing, etc.) after the base material layer forming step. For example, as a finishing step, a step of applying the skin layer resin on the release paper, a step of applying the adhesive resin on the surface of the skin layer resin opposite to the release paper, and the skin layer resin of the adhesive resin are A synthetic leather can be produced by a method including a step of stacking a base material layer on the opposite surface and heat-pressing. The synthetic leather produced by such a production method may be synthetic leather. FIG. 13 is a schematic view showing an example of a manufacturing apparatus for manufacturing synthetic leather. In the manufacturing method using the manufacturing apparatus 900 shown in FIG. 13, first, the paper pattern 91 is pulled out from the roll from which the paper pattern is wound, the skin layer resin (polymer) 92 is applied on the paper pattern 91, and the dryer 93 is used. Dry with. Next, the adhesive layer resin 94 is applied to the surface of the skin layer resin (polymer) 92 (the surface opposite to the surface in contact with the paper pattern 91) and dried by the dryer 93. Then, the base material layer 96 is pulled out from the roll around which the base material layer 96 is wound, and is laminated on the surface of the adhesive layer resin 94 (the surface opposite to the surface in contact with the skin layer resin (polymer) 92). A roll is made of synthetic leather 97 in which a base material layer 96, an adhesive layer resin 94, a skin layer resin (polymer) 92, and a release paper 91 are laminated in this order by thermocompression bonding with a hot press roll 95. By winding, synthetic leather can be manufactured.

The adhesive layer resin 94 is not particularly limited, and an adhesive used for conventional synthetic leather can be used. Examples of the adhesive include synthetic resins such as polyolefin, polystyrene, polyurethane, acrylic, and EVA (ethylene vinyl acetate copolymer). Examples of the skin layer resin (polymer) 92 include the above-mentioned polymer substances (polymer substances contained in the base material layer).

The thickness of the adhesive layer formed by the adhesive layer resin 94 is not particularly limited, but may be, for example, 1 to 500 μm, 10 to 400 μm, 50 to 300 μm, and 50 to 50. It may be 200 μm.

It should be noted that the synthetic leather according to the embodiment can be continuously or discontinuously manufactured without using the manufacturing apparatus or the like shown in FIGS. 12 or 13 or by using an apparatus different from those. .. For example, after immersing the entire non-woven fabric of a predetermined size in an impregnating liquid in an impregnating tank having an independent structure, the polymer substance impregnated in the non-woven fabric is coagulated with or without a coagulating liquid. .. Then, if necessary, the desired synthetic leather can be obtained by washing and drying.

If the non-woven fabric contains proteins, the non-woven fabric can be formed, for example, by electrospinning. In the electrospinning method (electrostatic spinning method), a voltage is applied between a supply side electrode (which can also be used as a spinneret) and a collection side electrode (for example, a metal roll or a metal net), and a dope liquid extruded from the spinneret. Is charged and blown off to the collecting side electrode. At this time, the doping liquid is stretched to form fibers. The applied voltage is usually 5 to 100 kV, preferably 10 to 50 kV. The distance between the electrodes is usually 1 to 25 cm, preferably 2 to 20 cm.

FIG. 14 is an explanatory diagram of the electrospinning device 100 according to the embodiment. A voltage is applied by the power supply 35 between the metal mouthpiece nozzle 33 (supply side electrode) and the metal net 38 (collection side electrode). The dope liquid 32 in the microsyringe 31 is moved in the direction of arrow P using a syringe pump, the dope liquid 32 is pushed out from the metal mouthpiece nozzle 33, and the dope liquid is expanded by electric charge to form a fibrous material 36 into a metal net 38. By accumulating on the surface of the above, a non-woven fabric 39 containing protein fibers can be obtained. The obtained non-woven fabric may then remove the solvent. Examples of the method for removing the solvent include drying under reduced pressure or immersion in a solvent removal tank.

(Use of synthetic leather)
The synthetic leather according to the present embodiment can be used, for example, for clothing, ornaments such as shoes and bags, various covers and furniture, and automobile interior materials.

<Artificial leather>
FIG. 8B is a schematic cross-sectional view of artificial leather. The base material layer is a base fabric layer 23 including a knitted fabric or a woven fabric. As the epidermis layer 25, the protein molded product according to the first embodiment can be used. The epidermis layer may be subjected to desired processing (dyeing, painting, graving, shrinking, embossing, etc.) so as to give an appearance closer to that of natural leather. If necessary, the adhesive layer 24 may be provided between the base material layer and the epidermis layer by a method well known to those skilled in the art.

(Base cloth layer)
The base fabric layer mainly contains a knitted fabric. The base fabric layer may be made of a knitted or woven material, but it does not exclude the inclusion of other components as long as the function as the base fabric layer is not impaired. In the synthetic leather according to this embodiment, the base cloth layer contains a protein. The base fabric layer may be composed of, for example, a knitted fabric containing protein fibers, or may be composed of a knitted fabric mixed with proteins (for example, a knitted fabric in which proteins are attached to a knitted fabric composed of synthetic fibers). It may have been done.

The knitted body is a general term for knitted fabrics and woven fabrics. The knitted fabric is a knitted fabric having a weft knitting structure such as a horizontal knitting or a circular knitting (also simply referred to as a "weft knitting fabric"), or a knitted fabric having a warp knitting structure such as tricot or Russell (also simply referred to as a "warp knitted fabric"). It may be any of.). The woven fabric may be a woven fabric having any of a plain weave, a twill weave, and a satin weave. The knitted or woven body may be an unprocessed knitted or woven body obtained by knitting or weaving, or may be a knitted or woven body that has been subjected to a process such as water repellent treatment after knitting or weaving.

The braided body can be obtained by knitting or weaving a raw material yarn. A known method can be used as the knitting method and the weaving method. As the knitting machine to be used, for example, a circular knitting machine, a warp knitting machine, a flat knitting machine and the like can be used, and from the viewpoint of productivity, the use of a circular knitting machine is preferable. Examples of the flat knitting machine include a molding knitting machine and a non-sewn knitting machine, but it is more preferable to use a non-sewn knitting machine because the knitted fabric can be manufactured in the form of a final product. Examples of the loom used include a shuttle loom and a non-shuttle loom such as a gripper loom, a rapier loom, and an air jet loom.

The raw material yarn may be a single yarn, a composite yarn (for example, a blended yarn, a mixed fiber yarn, a covering yarn, etc.), or a combination thereof may be used. The single yarn and the composite yarn may be spun yarn in which short fibers are twisted, or may be filament yarn in which long fibers are twisted. The fibers contained in the raw material yarn include, for example, synthetic fibers such as nylon, polyester, polyamide, polyacrylonitrile, polyolefin, polyvinyl alcohol, polyethylene terephthalate and polytetrafluoroethylene, recycled fibers such as cupra, rayon and lyocell, cotton and hemp. And natural fibers such as silk, and protein fibers.

The braided body preferably contains protein fibers. The protein fiber preferably contains artificial fibroin, and more preferably contains artificial spider silk fibroin. By containing artificial fibroin (preferably artificial spider silk fibroin), it is possible to impart heat retention, hygroscopic heat generation and / or flame retardant functionality to the braided body. As a result, the synthetic leather according to the present embodiment can be imparted with heat retention, hygroscopic heat generation and / or flame retardant functionality, and its value as a material is further increased. The artificial fibroin may be contained in the braided body as artificial fibroin fibers (protein fibers). Preferred embodiments of artificial fibroin will be described later.

The base fabric layer may be a single layer or may have a multi-layer structure composed of a plurality of knitted bodies. The thickness of the base fabric layer is not particularly limited, but may be, for example, 100 to 2000 μm, 100 to 1000 μm, 500 to 1000 μm, or 500 to 750 μm.

(Epidermis layer)
The epidermis layer is a layer mainly formed of a resin (polymer). The epidermis layer may be formed of a porous resin or a non-porous resin. The layer formed of the porous resin (porous layer) can be formed, for example, by producing synthetic leather by a wet method described later.

Examples of the polymer forming the epidermis layer include synthetic resins such as polyvinyl chloride, polyolefin, polystyrene, polyurethane, polyester resin, epoxy resin, acrylic polymer, and acrylonitrile polymer, and proteins. As the resin (polymer) forming the epidermis layer, a composite material of a synthetic resin and a protein may be used. Examples of the polyurethane include polyether polyurethane, polyester polyurethane, and polycarbonate polyurethane, and these may be used alone or in combination of two or more.

The epidermis layer preferably contains a protein. The protein preferably contains artificial fibroin, and more preferably contains artificial spider silk fibroin. By containing artificial fibroin (preferably artificial spider silk fibroin), it is possible to impart heat retention, hygroscopic heat generation and / or flame retardant functionality to the epidermis layer. As a result, the synthetic leather according to the present embodiment can be imparted with heat retention, hygroscopic heat generation and / or flame retardant functionality, and its value as a material is further increased. Preferred embodiments of artificial fibroin will be described later.

The epidermis layer may contain known additives, if desired. Examples of the additive include a colorant, a smoothing agent, an antioxidant, an ultraviolet absorber, a dye, a filler, a cross-linking agent, a matting agent, a leveling agent and the like.

The thickness of the epidermis layer is not particularly limited, but may be, for example, 1 to 500 μm, 1 to 400 μm, 5 to 300 μm, or 50 to 200 μm.

The surface of the epidermis layer (the surface opposite to the surface bonded to the base fabric layer) may be surface-treated. A known method can be applied to the surface processing. Examples of the surface processing include embossing, buff suede processing, film laminating processing, and gravure processing (gravure printing).

(Manufacturing method of artificial leather)
The artificial leather according to the present embodiment can be produced according to a conventional method except that a base fabric layer (knitted body) containing a protein is used. The artificial leather according to the present embodiment can be produced, for example, by a method including a step of forming a skin layer on a base cloth layer including a knitted body. The artificial leather according to the present embodiment can be produced by, for example, either a dry method or a wet method.

In the dry method, for example, the step of applying the skin layer resin on the release paper, the step of applying the adhesive resin on the surface of the skin layer resin opposite to the release paper, and the skin layer resin of the adhesive resin are Artificial leather can be manufactured by a method including a step of stacking a base cloth (knitted body) on the opposite surface and thermocompression bonding. FIG. 15 is a schematic view showing an example of a manufacturing apparatus for manufacturing artificial leather by a dry method. In the manufacturing method using the manufacturing apparatus 100 shown in FIG. 15, first, the paper pattern 50 is pulled out from the roll on which the paper pattern is wound, the skin layer resin (polymer) 51 is applied on the paper pattern 50, and the dryer 60 is used. Dry with. Next, the adhesive layer resin 52 is applied to the surface of the skin layer resin (polymer) 51 (the surface opposite to the surface in contact with the paper pattern 50) and dried in the dryer 60. Then, the knitted fabric (base cloth) 53 is pulled out from the roll on which the knitted fabric is wound, and is laminated on the surface of the adhesive layer resin 52 (the surface opposite to the surface in contact with the skin layer resin (polymer) 51). , Artificial leather in which the braided body (base cloth) 53, the adhesive layer resin 52, the skin layer resin (polymer) 51, and the release paper 50 are laminated in this order by thermocompression bonding with a hot press roll 70. Artificial leather can be manufactured by winding 54 into a roll. If the skin layer resin can be integrally bonded to the base cloth by, for example, thermocompression bonding, depending on the type of the skin layer resin and the material that gives the base cloth, the adhesive layer resin may be omitted. Is also possible.

The adhesive layer resin 52 is not particularly limited, and an adhesive used in conventional artificial leather can be used. Examples of the adhesive include synthetic resins such as polyolefin, polystyrene, polyurethane, acrylic, and ethylene-vinyl acetate copolymer (EVA).

The thickness of the adhesive layer formed by the adhesive layer resin 52 is not particularly limited, but may be, for example, 1 to 50 μm, 1 to 40 μm, 5 to 30 μm, 5 to 30 μm. It may be 20 μm.

In the wet method, for example, a step of applying a liquid containing a skin layer resin on a base cloth (knitted body) and a step of immersing the base cloth coated with the liquid containing the skin layer resin in a coagulating liquid to form a skin layer. Artificial leather can be manufactured by the method provided with and. FIG. 16 is a schematic view showing an example of a manufacturing apparatus for manufacturing artificial leather by a wet method. In the manufacturing method using the manufacturing apparatus 200 shown in FIG. 16, first, the knitted fabric (base cloth) 53 is pulled out from the roll from which the knitted fabric is wound, and the skin layer resin (polymer) is drawn from the coating head 55. Cloth) 53 is applied on top. Next, the skin layer resin (polymer) is solidified in the coagulation tank 80 containing the coagulation liquid to form the skin layer on the knitted body (base cloth) 53. Then, after washing through a hot water washing tank 90 containing a washing liquid, the artificial leather is dried by a dryer 60, and the artificial leather 54 having the skin layer bonded on the knitted body (base cloth) 53 is wound on a roll to obtain the artificial leather. Can be manufactured. In the wet method, it is also possible to form a porous epidermis layer. For example, the base cloth is coated with a solvent solution mainly composed of a polymer resin as a skin layer resin. Then, for example, when immersed in a coagulation liquid mainly composed of water, the solvent in the skin layer resin elutes into the coagulation liquid, and at the same time, the coagulation liquid mainly composed of water permeates and diffuses into the skin layer resin, and the skin layer resin. Is wet solidified. As a result, a porous epidermis layer can be formed. Depending on the type of solvent of the solution containing the epidermis layer resin (for example, one having a low boiling point), the base cloth coated with the solution containing the epidermis layer resin contains the epidermis layer resin without contacting with the coagulating liquid. The resin for the epidermis layer may be coagulated to form the epidermis layer by removing the solvent by volatilizing the solvent of the solution.

The type of coagulation liquid can be appropriately selected depending on the type of the epidermis layer resin (polymer) and the solvent that dissolves or disperses the epidermis layer resin. For example, when the epidermis layer resin (polymer) is polyurethane, for example, dimethylformamide can be used as the solvent and water (hot water) can be used as the coagulation liquid. Further, for example, when the epidermis resin (polymer) is a protein, for example, dimethylsulfoxide (DMSO), N, N-dimethylformamide (DMF), formic acid, and hexafluoroisopropanol (HFIP) as solvents and dissolved in these. A product to which an inorganic salt is added as an accelerator, a lower alcohol having 1 to 5 carbon atoms such as methanol, ethanol and 2-propanol, acetone, and water can be used as the coagulation liquid. Various additives may be added to the coagulation liquid as needed.

It should be noted that the synthetic leather according to the present embodiment can be manufactured discontinuously without using the manufacturing apparatus or the like shown in FIG. 15 or FIG. 16 or by using an apparatus other than those. For example, a laminated skin layer resin is applied to the surface of a base cloth having a predetermined size that has not been wound, and the skin layer resin is coagulated with or without a coagulating liquid. Then, if necessary, the desired synthetic leather can be obtained by washing and drying.

(Use of artificial leather)
The synthetic leather according to the present embodiment can be used for applications in which conventional artificial leather (for example, synthetic leather composed of synthetic resin) has been used. The artificial leather according to the present embodiment can be used, for example, for clothing, ornaments such as shoes and bags, various covers and furniture, and automobile interior materials.

Specific embodiments according to the present embodiment include the following.

Embodiment 1 comprises a substrate layer and an epidermis layer bonded onto the substrate layer, wherein the epidermis layer is formed by a linker whose protein is represented by the formula (1), (2) or (3). An artificial leather containing a molded product containing a crosslinked crosslinked protein. The base material layer is preferably a base fabric containing any of a knitted fabric, a woven fabric, and a non-woven fabric.

The second embodiment is a method for producing artificial leather including a base material layer and a skin layer bonded on the base material layer, wherein the protein and the formulas (1a), (2a), (2b) or ( The skin layer comprises a step of spreading a composition containing the compound represented by 3a) to form a skin layer precursor and a step of heating the skin layer precursor, and the skin layer comprises the protein. It is a method for producing artificial leather containing a crosslinked protein crosslinked by a linker represented by the formula (1), (2) or (3).

The third embodiment is a method for producing an artificial leather including a base material layer and a skin layer bonded on the base material layer, in which protein fibers containing a crosslinked protein are knitted or woven to obtain the base material layer. The cross-linked protein is a method for producing artificial leather, which comprises a step and the protein is cross-linked by a linker represented by the formula (1), (2) or (3).

In the fourth embodiment, a base material is provided with a base material layer and a skin layer bonded onto the base material layer, the base material layer is a base cloth containing a knitted cloth or a woven cloth, and the base cloth is a base cloth containing a protein. An artificial leather containing a molded product containing a fibrous crosslinked protein crosslinked by a linker represented by the formula (1), (2) or (3).

Embodiment 5 comprises a substrate layer comprising a non-woven fabric impregnated with a polymer composition, wherein the non-woven fabric is a fiber in which the protein is crosslinked by a linker represented by the formula (1), (2) or (3). Artificial leather containing a molded product containing a cross-linked protein in the form.

Embodiment 6 comprises a substrate layer comprising a non-woven fabric impregnated with the polymer composition, wherein the polymer composition is comprising a polymer and a linker in which the protein is represented by the formula (1), (2) or (3). An artificial leather containing a molded product containing a crosslinked protein.

Embodiment 7 is a method for producing artificial leather provided with a base material layer, which comprises a step of impregnating a non-woven fabric with a polymer composition containing a crosslinked protein to obtain a base layer, wherein the crosslinked protein has a protein formula ( It is a method for producing artificial leather, which is crosslinked by a linker represented by 1), (2) or (3).

Embodiment 8 is a method for producing artificial leather having a base material layer, which comprises a composition containing a protein and a compound represented by the formulas (1a), (2a), (2b) or (3a). A step of obtaining a base layer by heating while impregnating the non-woven fabric is included, and the base layer is a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1), (2) or (3). It is a method for producing artificial leather, which comprises a non-woven fabric impregnated with a molded product containing.

[Manufacturing of artificial fibroin]
(1) Preparation of expression vector An artificial fibroin (PRT966) having the amino acid sequence shown in SEQ ID NO: 40 was designed. Nucleic acids encoding the designed artificial fibroin were synthesized. An NdeI site was added to the nucleic acid at the 5'end, and an EcoRI site was added downstream of the stop codon. This nucleic acid was cloned into a cloning vector (pUC118). Then, the nucleic acid was cut out by restriction enzyme treatment with NdeI and EcoRI, and then recombinant into a protein expression vector pET-22b (+) to obtain an expression vector.

(2) Expression of protein Escherichia coli BLR (DE3) was transformed with the obtained expression vector. The transformed E. coli was cultured in 2 mL of LB medium containing ampicillin for 15 hours. The culture solution was added to 100 mL of seed culture medium (Table 4) containing ampicillin so that OD _{600 was 0.005.} The culture solution temperature was maintained at 30 ° C., _{and flask culture was carried out until the OD 600} reached 5, (about 15 hours) to obtain a seed culture solution.

The seed culture solution was added to a jar fermenter to which 500 mL of the production medium (Table 5) was added so that the OD ₆₀₀ was 0.05. The culture solution temperature was maintained at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration.

Immediately after the glucose in the production medium was completely consumed, the feed solution (glucose 455 g / 1 L, Yeast Extract 120 g / 1 L) was added at a rate of 1 mL / min. The culture solution temperature was maintained at 37 ° C., and the culture was controlled at a constant pH of 6.9. Further, the dissolved oxygen concentration in the culture solution was maintained at 20% of the dissolved oxygen saturation concentration, and the culture was carried out for 20 hours. Then, 1 M of isopropyl-β-thiogalactopyranoside (IPTG) was added to the culture solution to a final concentration of 1 mM to induce the expression of artificial fibroin. Twenty hours after the addition of IPTG, the culture solution was centrifuged and the cells were collected. SDS-PAGE was performed using cells prepared from the culture medium before and after the addition of IPTG, and the expression of the target artificial fibroin was confirmed by the appearance of the target artificial fibroin-sized band depending on the IPTG addition. bottom.

(3) Purification of protein The cells collected 2 hours after the addition of IPTG were washed with 20 mM Tris-HCl buffer (pH 7.4). The washed cells were suspended in 20 mM Tris-HCl buffer (pH 7.4) containing about 1 mM PMSF, and the cells were disrupted with a high-pressure homogenizer (manufactured by GEA Niro Soavi). The crushed cells were centrifuged to obtain a precipitate. The resulting precipitate was washed with 20 mM Tris-HCl buffer (pH 7.4) until high purity. The washed precipitate was suspended in 8M guanidine buffer (8M guanidine hydrochloride, 10 mM sodium dihydrogen phosphate, 20 mM NaCl, 1 mM Tris-HCl, pH 7.0) to a concentration of 100 mg / mL and at 60 ° C. Stir with a stirrer for 30 minutes to dissolve. After dissolution, dialysis was performed with water using a dialysis tube (cellulose tube 36/32 manufactured by Sanko Junyaku Co., Ltd.). The white aggregated protein obtained after dialysis was recovered by centrifugation, water was removed by a freeze-dryer, and the freeze-dried powder was recovered to obtain artificial fibroin (PRT966).

<Example 1>
Artificial spider fibroin (SEQ ID NO: 40) 7.68 g and 4,4'-bismaleimideimide diphenylmethane (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.32 g, attritor crusher (manufactured by Nippon Coke Industries Co., Ltd., PWB 0018 model) Was pulverized and mixed at a rate of 1000 rpm for 10 minutes to obtain a mixed powder (added with 4 wt% maleimide). The obtained mixed powder was put into a mold, and a molded product was obtained by heating and pressurizing under the conditions of a temperature of 160 ° C., a pressure of 50 MPa, and a holding time of 3 minutes.

<Example 2>
Artificial spider fibroin (SEQ ID NO: 40) 4.9 g and 4,4'-bismaleimideimide diphenylmethane (manufactured by Tokyo Chemical Industry Co., Ltd.) 0.1 g, attritor crusher (manufactured by Nippon Coke Industries Co., Ltd., PWB 0018 model) Was pulverized and mixed at a rate of 1000 rpm for 10 minutes to obtain a mixed powder (added with 2 wt% maleimide). The obtained mixed powder was put into a mold, and a molded product was obtained by heating and pressurizing under the conditions of a temperature of 160 ° C., a pressure of 30 MPa, and a holding time of 3 minutes.

<Comparative example 1>
8 g of artificial spider silk fibroin (SEQ ID NO: 40) was pulverized and mixed at a speed of 1000 rpm for 10 minutes using an attritor crusher (manufactured by Nippon Coke Industries, Ltd., PWB 0018 model) to obtain a powder. The obtained powder was put into a mold, and a molded product was obtained by heating and pressurizing under the conditions of a temperature of 160 ° C., a pressure of 50 MPa, and a holding time of 3 minutes.

Test 1: Bending test (1) Evaluation Each molded product of Example 1 and Comparative Example 1 was subjected to a precision universal testing machine (Autograph AG-XD plus, 50 kN load cell manufactured by Shimadzu Corporation) at a speed of 1 mm / min, and a branch office. A three-point bending test was carried out under the conditions of an interval of 28 mm and n = 5.

(2) Results The results are shown in Table 6. Example 1 and Comparative Example 1 showed similar strength and elastic modulus to each other. In addition, Table 6 shows the strength and elastic modulus of Example 1 as the ratio to Comparative Example 1 when the strength and elastic modulus of Comparative Example 1 were set to 100, respectively.

Test 2: GPC test (1) Evaluation After crushing the molded products of Example 1 and Comparative Example 1, GPC (gel permeation chromatography) measurement was performed to measure the molecular weight of crosslinked artificial fibroin in each molded product.

(2) Results The results are shown in Table 7 and FIG. The peak of Example 1 had a shorter elution time than that of Comparative Example 1, suggesting that the molecular weight increased due to the cross-linking reaction. Moreover, the PDI value of Example 1 was higher than that of Comparative Example 1. The cross-linking reaction suggests that Example 1 has increased side chain variations due to the longer side chains. From the above, it was found that when the mixed powder of Example 1 was heated and pressed, cross-linking proceeded at the same time as molding.

3. 3. Crack test (1) Evaluation Using the molded products of Example 1 and Comparative Example 1, a crack test was carried out according to the following procedure. Both molded products were stored in a constant temperature and humidity chamber (manufactured by Etac Co., Ltd., trade name: Highflex FX410N) under the conditions of a temperature of 20 ° C. and a humidity of 65% RH for 2 weeks. Then, both molded bodies were transferred into a dryer (manufactured by AS ONE, trade name: ETTAS AVO-250NS-D) and stored at a temperature of 50 ° C. for 140 hours. Further, both molded products were stored at a temperature of 80 ° C. for 192 hours. The appearance of both molded bodies was evaluated over time.

(2) Results The results are shown in FIG. In Comparative Example 1, cracks were observed when stored at 50 ° C. for 1.5 hours, whereas in Example 1, cracks occurred even after storage at 80 ° C. for 192 hours. There wasn't. From this result as well, the heating and pressurizing of the mixed powder of Example 1 sufficiently promotes cross-linking at the same time as molding the resin, thereby suppressing hygroscopicity and suppressing the occurrence of cracks in a high humidity environment. It turned out that it could be done.

4. Hygroscopicity test (1) Evaluation The molded bodies (n = 3 each) of Example 2 and Comparative Example 1 were subjected to a constant temperature and humidity chamber (Lightspec constant temperature and humidity chamber manufactured by ESPEC, trade name: LHL-114) at a temperature of 30. The sample was stored for 43 days under the conditions of ° C. and humidity of 93% RH, and the mass change of the sample before and after the test was measured.

(2) Results The results are shown in FIG. The water absorption rate of the molded product of Comparative Example 1 was 7.6%, whereas the water absorption rate of the molded product of Example 2 was 4.1%. It is considered that this is because the cross-linking effect suppressed the hygroscopicity of the molded product of Example 2 under constant humidity conditions.

2 ... Mold, 4 ... Upper pin, 6 ... Lower pin, 8a ... Composition before heating and pressurizing, 8b ... Composition after heating and pressurizing, 10 ... Pressure molding machine, 11 ... Non-woven fabric, 12 ... Artificial fibroin multimer, 13, 23 ... Base layer, 14, 24 ... Adhesive layer (optional), 15, 25 ... Skin layer.

Claims (20)

A molded product of a composition containing a crosslinked protein.
A molded product in which the crosslinked protein is a protein crosslinked by a linker represented by the formula (1), (2) or (3).

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ] The molded product according to claim 1, wherein the protein is an artificial protein. The molded product according to claim 2, wherein the artificial protein is an artificial structural protein. The molded product according to claim 3, wherein the artificial structural protein is artificial fibroin. The molded product according to claim 4, wherein the artificial fibroin is artificial spider silk fibroin. The molded product according to any one of claims 1 to 5, which is in the form of a film or a fiber. The molded product according to any one of claims 1 to 6, which is apparently a uniform phase. A method for producing a molded product of a composition containing a crosslinked protein.
A method for producing a molded product, which comprises a step of heating a composition containing a protein and a compound represented by the formula (1a), (2a), (2b) or (3a).

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ] The molded product is in the form of fibers.
The method for producing a molded product according to claim 8, wherein the heating is heating for drying undried fibers obtained by dry spinning, wet spinning, or dry-wet spinning. The molded product is in the form of a film.
The method for producing a molded product according to claim 8, wherein the heating is heating for drying an undried film obtained by cast molding or for an annealing treatment. The method for producing a molded product according to claim 8, wherein the composition is pressurized at the same time as heating. The method for producing a molded product according to any one of claims 8 to 11, wherein the molded product has an apparently uniform phase. A base material layer and an epidermis layer bonded onto the base material layer are provided.
Artificial leather in which the epidermis layer contains a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1), (2) or (3).

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ] A base material layer and an epidermis layer bonded onto the base material layer are provided.
The base material layer is a base fabric containing a knitted fabric or a woven fabric.
An artificial leather in which the base fabric contains a molded product containing a fibrous crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1), (2) or (3).

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ] It comprises a substrate layer containing a non-woven fabric impregnated with a polymer composition.
An artificial leather in which the non-woven fabric contains a molded product containing a fibrous crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1), (2) or (3).

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ] It comprises a substrate layer containing a non-woven fabric impregnated with a polymer composition.
An artificial leather in which the polymer composition contains a molded product containing a crosslinked protein in which the protein is crosslinked by a linker represented by the formula (1), (2) or (3).

[In the formula, L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ]

[In the formula, R represents a hydrogen atom or an organic group, and L represents a divalent organic group. ] The artificial leather according to any one of claims 13 to 16, wherein the protein is an artificial protein. The artificial leather according to claim 17, wherein the artificial protein is an artificial structural protein. The artificial leather according to claim 18, wherein the artificial structural protein is artificial fibroin. The artificial leather according to claim 19, wherein the artificial fibroin is artificial spider silk fibroin.

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